Integrated Continuous Manufacturing of Therapeutic Protein Drug Substances

ABSTRACT

Provided herein are integrated continuous biomanufacturing processes for producing a therapeutic protein drug substance. Also provided are systems that are capable of continuously producing a therapeutic protein drug substance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/775,060, filed Mar. 8, 2013, and U.S. Provisional PatentApplication No. 61/856,390, filed Jul. 19, 2013, the entire contents ofthese two applications are herein incorporated by reference.

TECHNICAL FIELD

This invention relates to methods of biotechnology and thebiomanufacturing of recombinant proteins.

BACKGROUND

Mammalian cells containing a nucleic acid that encodes a recombinantprotein are often used to produce therapeutically or commerciallyimportant proteins. In the current environment of diverse productpipelines, biotechnology companies are increasingly driven to developinnovative solutions for highly flexible and cost-effectivemanufacturing of therapeutic protein drug substances.

SUMMARY

The present invention is based, at least in part, on the discovery thatintegrated, continuous systems that include two multi-columnchromatography systems can be used to continuously produce therapeuticprotein drug substances. In view of this discovery, provided herein areintegrated and continuous processes for manufacturing a therapeuticprotein drug substance that include providing a liquid culture mediumcontaining a recombinant therapeutic protein that is substantially freeof cells, where the liquid culture medium is fed into a firstmulti-column chromatography system (MCCS1); capturing the recombinanttherapeutic protein in the liquid culture medium using the MCCS1, wherethe eluate of the MCCS1 containing the recombinant therapeutic proteinis continuously fed into a second multi-column chromatography system(MCCS2); and purifying and polishing the recombinant therapeutic proteinusing the MCCS2, where the eluate from the MCCS2 is a therapeuticprotein drug substance; and where the process is integrated and runscontinuously from the liquid culture medium to the eluate from the MCCS2that is the therapeutic protein drug substance. Also provided aresystems specifically designed to perform any of the processes describedherein. For example, provided herein are biological manufacturingsystems that include: a first multi-column chromatography system (MCCS)containing an inlet; and a second MCCS including an outlet, where thefirst and second MCCSs are in fluid communication with each other, andwhere the manufacturing system is configured such that fluid can bepassed into the inlet, through the first and second MCCSs, and exit themanufacturing system through the outlet.

Provided herein are integrated and continuous processes formanufacturing a therapeutic protein drug substance that include: (i)providing a liquid culture medium containing a recombinant therapeuticprotein that is substantially free of cells, where the liquid culturemedium is fed into a first multi-column chromatography system (MCCS1);(ii) capturing the recombinant therapeutic protein in the liquid culturemedium using the MCCS1, where the eluate of the MCCS1 containing therecombinant therapeutic protein is continuously fed into a secondmulti-column chromatography system (MCCS2); and (iii) purifying andpolishing the recombinant therapeutic protein using the MCCS2, where theeluate from the MCCS2 is a therapeutic protein drug substance; and wherethe process is integrated and runs continuously from the liquid culturemedium to the eluate from the MCCS2 that is the therapeutic protein drugsubstance. In some embodiments of any of the processes described herein,the liquid culture medium is selected from the group of: liquid culturemedium removed from a perfusion bioreactor containing a culture ofmammalian cells that secrete the recombinant therapeutic protein, liquidculture medium removed from a fed-batch bioreactor containing a cultureof mammalian cells that secrete the recombinant therapeutic protein, anda clarified liquid culture medium from a culture of bacteria or yeastcells that secrete the recombinant therapeutic protein.

In some embodiments of any of the processes described herein, the MCCS1and/or the MCCS2 performs at least two different unit operations. Insome embodiments of any of the processes described herein, the use ofthe MCCS1 or the MCCS2, or both, involves column switching. In someembodiments of any of the processes described herein, the MCCS1 performsthe unit operations of capturing the recombinant therapeutic protein andinactivating viruses. In some embodiments of any of the processesdescribed herein, the MCCS2 performs the unit operations of purifyingand polishing the recombinant therapeutic protein. In some embodimentsof any of the processes described herein, the MCCS1 and/or the MCCS2utilizes at least two chromatography columns. In some embodiments of anyof the processes described herein, the MCCS1 and/or the MCCS2 utilizesat least two chromatographic membranes. In some embodiments of any ofthe processes described herein, the MCCS1 and/or the MCCS2 utilizes atleast one chromatography column and at least one chromatographicmembrane. In some embodiments of any of the processes described herein,the liquid culture medium is liquid culture medium removed from aperfusion bioreactor containing a culture of mammalian cells thatsecrete the recombinant therapeutic protein.

In some embodiments of any of the processes described herein, the MCCS1is a first periodic counter current chromatography system (PCCS1). Insome embodiments of any of the processes described herein, the PCCS1includes a four-column PCCS. In some embodiments of any of the processesdescribed herein, three of the four columns in the four-column PCCSperform the unit operation of capturing the recombinant therapeuticprotein from the liquid culture medium. In some embodiments of any ofthe processes described herein, the capturing is performed usingaffinity chromatography, cation exchange chromatography, anion exchangechromatography, or molecular sieve chromatography. In some embodimentsof any of the processes described herein, the affinity chromatography isperformed with a capture mechanism selected from the group of: proteinA-binding capture mechanism, substrate-binding capture mechanism,antibody- or antibody fragment-binding capture mechanism,aptamer-binding capture mechanism, and cofactor-binding capturemechanism. In some embodiments of any of the processes described herein,the affinity chromatography is performed with a protein-A bindingcapture mechanism, and the recombinant therapeutic protein is anantibody or an antibody fragment. In some embodiments of any of theprocesses described herein, the eluate containing the recombinanttherapeutic protein from the three of the four columns in thefour-column PCCS is fed into the fourth column of the four-column PCCS.In some embodiments of any of the processes described herein, the fourthcolumn of the four-column PCCS performs the unit operation ofinactivating viruses by holding the eluate containing recombinanttherapeutic protein at a low pH for viral inactivation. In someembodiments of any of the processes described herein, the fourth columnof the four-column PCCS holds the eluate containing the recombinanttherapeutic protein at a low pH for viral inactivation for a period ofabout 10 minutes to about 1.5 hours.

In some embodiments of any of the processes described herein, the MCCS2is a second periodic counter current (PCCS2) chromatography system. Someembodiments of any of the processes described herein further includeadjusting the pH of the eluate from the fourth column of the four-columnPCCS using an in-line buffer adjustment reservoir before the eluate fromthe fourth column of the four-column PCCS is fed into the PCCS2. In someembodiments of any of the processes described herein, the PCCS2chromatography system includes a three chromatography columns and achromatographic membrane. In some embodiments of any of the processesdescribed herein, the three chromatography columns in the PCCS2 performthe unit operation of purifying the recombinant therapeutic protein fromthe eluate of the PCCS1 through cation or anion exchange chromatography.In some embodiments of any of the processes described herein, the eluatefrom the three chromatography columns in the PCCS2 is fed into thechromatographic membrane in the PCCS2. In some embodiments of any of theprocesses described herein, the chromatographic membrane in the PCCS2performs the unit function of polishing the recombinant therapeuticprotein present in the eluate from the three chromatography columns inthe PCCS2 through cation or anion exchange chromatography. In someembodiments of any of the processes described herein, thechromatographic membrane in the PCCS2 performs the unit function ofpolishing through cation exchange chromatography. In some embodiments ofany of the processes described herein, the flow through and wash of thechromatographic membrane is the therapeutic protein drug substance. Someembodiments of any of the processes described herein further includeformulating the therapeutic protein drug substance into a pharmaceuticalcomposition.

In some embodiments of any of the processes described herein, therecombinant therapeutic protein is an antibody or antibody fragment, anenzyme, an engineered protein, or an immunogenic protein or proteinfragment. Some embodiments of any of the processes described hereinfurther include adjusting the ionic concentration of the eluate from thethree columns in the PCCS2 using in-line buffer adjustment before theeluate from the three columns in the PCCS2 is fed into thechromatographic membrane in the PCCS2. Some embodiments of any of theprocesses described herein further include the use of a break tank(e.g., any break tank described herein) between the PCCS1 and the PCCS2.Some embodiments of any of the processes described herein furtherinclude filtering the eluate from the PCCS1 before it is fed into thePCCS2. Some embodiments of any of the methods described herein furtherinclude filtering the liquid culture medium before it is fed into theMCCS1.

Also provided are integrated and continuous processes for manufacturinga therapeutic protein drug substance that include: (i) culturingmammalian cells that secrete a recombinant therapeutic protein in aperfusion bioreactor that contains a liquid culture medium, where avolume of the liquid culture medium that is substantially free of cellsis continuously or periodically removed from the perfusion bioreactorand fed into a first multi-column chromatography system (MCCS1); (ii)capturing the recombinant therapeutic protein in the removed liquidculture medium using the MCCS1, where the eluate of the MCCS1 containingthe recombinant therapeutic protein is continuously fed into a secondmulti-column chromatography system (MCCS2); and (iii) purifying andpolishing the therapeutic recombinant protein in the eluate of the MCCS1using the MCCS2, where the eluate from the MCCS2 is a therapeuticprotein drug substance; where the process is integrated and runscontinuously from the removed liquid culture medium to the eluate fromthe MCCS2 that is the therapeutic protein drug substance.

In some embodiments of any of the processes described herein, the MCCS1and/or the MCCS2 performs at least two different unit operations. Insome embodiments of any of the processes described herein, the use ofthe MCCS1 or the MCCS2, or both, involves column switching. In someembodiments of any of the processes described herein, the MCCS1 performsthe unit operations of capturing the recombinant therapeutic protein andinactivating viruses. In some embodiments of any of the processesdescribed herein, the MCCS2 performs the unit operations of purifyingand polishing the recombinant therapeutic protein. In some embodimentsof any of the processes described herein, the MCCS1 and/or the MCCS2utilizes at least two chromatography columns. In some embodiments of anyof the processes described herein, the MCCS1 and/or the MCCS2 utilizesat least two chromatographic membranes. In some embodiments of any ofthe processes described herein, the MCCS1 and/or the MCCS2 utilizes atleast one chromatography column and at least one chromatographicmembrane.

In some embodiments of any of the processes described herein, the MCCS1is a first periodic counter current chromatography system (PCCS1). Insome embodiments of any of the processes described herein, the PCCS1includes a four-column PCCS. In some embodiments of any of the processesdescribed herein, three of the four columns in the four-column PCCSperform the unit operation of capturing the recombinant therapeuticprotein from the liquid culture medium. In some embodiments of any ofthe processes described herein, the capturing is performed usingaffinity chromatography, cation exchange chromatography, anion exchangechromatography, or molecular sieve chromatography. In some embodimentsof any of the processes described herein, the affinity chromatography isperformed with a capture mechanism selected from the group of: proteinA-binding capture mechanism, substrate-binding capture mechanism,antibody- or antibody fragment-binding capture mechanism,aptamer-binding capture mechanism, and cofactor-binding capturemechanism. In some embodiments of any of the processes described herein,the affinity chromatography is performed with a protein-A bindingcapture mechanism, and the recombinant therapeutic protein is anantibody or an antibody fragment. In some embodiments of any of theprocesses described herein, the eluate containing the recombinanttherapeutic protein from the three of the four columns in thefour-column PCCS is fed into the fourth column of the four-column PCCS.In some embodiments of any of the processes described herein, the fourthcolumn of the four-column PCCS performs the unit operation ofinactivating viruses by holding the eluate containing recombinanttherapeutic protein at a low pH for viral inactivation. In someembodiments of any of the processes described herein, the fourth columnof the four-column PCCS holds the eluate containing the recombinanttherapeutic protein at a low pH for viral inactivation for a period ofabout 10 minutes to about 1.5 hours.

In some embodiments of any of the processes described herein, the MCCS2is a second periodic counter current (PCCS2) chromatography system. Someembodiments of any of the processes described herein further includeadjusting the pH of the eluate from the fourth column of the four-columnPCCS using an in-line buffer adjustment reservoir before the eluate fromthe fourth column of the four-column PCCS is fed into the PCCS2. In someembodiments of any of the processes described herein, the PCCS2chromatography system includes a three chromatography columns and achromatographic membrane. In some embodiments of any of the processesdescribed herein, the three chromatography columns in the PCCS2 performthe unit operation of purifying the recombinant therapeutic protein fromthe eluate of the PCCS1 through cation or anion exchange chromatography.In some embodiments of any of the processes described herein, the eluatefrom the three chromatography columns in the PCCS2 is fed into thechromatographic membrane in the PCCS2. In some embodiments of any of theprocesses described herein, the chromatographic membrane in the PCCS2performs the unit function of polishing the recombinant therapeuticprotein present in the eluate from the three chromatography columns inthe PCCS2 through cation or anion exchange chromatography. In someembodiments of any of the processes described herein, thechromatographic membrane in the PCCS2 performs the unit function ofpolishing through cation exchange chromatography. In some embodiments ofany of the processes described herein, the flow through and wash of thechromatographic membrane is the therapeutic protein drug substance. Someembodiments of any of the processes described herein further includeformulating the therapeutic protein drug substance into a pharmaceuticalcomposition. In some embodiments of any of the processes describedherein, the recombinant therapeutic protein is an antibody or antibodyfragment, an enzyme, an engineered protein, or an immunogenic protein orprotein fragment.

Some embodiments of any of the methods described herein further includeadjusting the ionic concentration of the eluate from the three columnsin the PCCS2 using in-line buffer adjustment before the eluate from thethree columns in the PCCS2 is fed into the chromatographic membrane inthe PCCS2. Some embodiments of any of the processes described hereinfurther include the use of a break tank (e.g., any break tank describedherein) between the PCCS1 and the PCCS2. Some embodiments of any of theprocesses described herein further include filtering the eluate from thePCCS1 before it is fed into the PCCS2. Some embodiments of any of theprocesses described herein further include filtering the liquid culturemedium before it is fed into the MCCS1.

Also provided are biological manufacturing system that include: a firstmulti-column chromatography system (MCCS) including an inlet; and asecond MCCS including an outlet, where the first and second MCCSs are influid communication with each other, and where the manufacturing systemis configured such that fluid can be passed into the inlet, through thefirst and second MCCSs, and exit the manufacturing system through theoutlet. Some embodiments of any of the systems described herein furtherinclude a bioreactor, where the bioreactor and the inlet are in fluidcommunication with each other, and where the manufacturing system isconfigured such that fluid present in the bioreactor can be passed intothe inlet. In some embodiments of any of the systems described herein,the first MCCS or the second MCCS, or both, is/are configured to performat least two separate unit operations. In some embodiments of any of thesystems described herein, use of the MCCS1 or the MCCS2, or both,involves column switching.

In some embodiments of any of the systems described herein, the firstMCCS is configured to perform the unit operations of capturing therecombinant therapeutic protein and inactivating viruses. In someembodiments of any of the systems described herein, the second MCCS isconfigured to perform the unit operations of purifying and polishing therecombinant therapeutic protein. In some embodiments of any of thesystems described herein, the first MCCS or the second MCCS, or both,contain(s) at least two chromatography columns. In some embodiments ofany of the systems described herein, the first MCCS or the second MCCS,or both, contain(s) at least two chromatographic membranes. In someembodiments of any of the systems described herein, the first MCCS orthe second MCCS, or both, contain(s) at least one chromatography columnand at least one chromatographic membrane.

In some embodiments of any of the systems described herein, the firstMCCS is a first periodic counter current chromatography system (PCCS1).In some embodiments of any of the systems described herein, the PCCS1includes a four-column PCCS. In some embodiments of any of the systemsdescribed herein, three of the four columns in the four-column PCCS arecapable of capturing the recombinant therapeutic protein from the liquidculture medium. In some embodiments of any of the systems describedherein, the three of the four columns in the four-column PCCS includeone or more of an affinity chromatography column, a cationicchromatography column, an anionic chromatography column, and a molecularsieve chromatography column. In some embodiments of any of the systemsdescribed herein, the three of the four columns in the four-column PCCSinclude one or more of an affinity chromatography column utilizing acapture mechanism selected from the group of: protein A-binding capturemechanism, substrate-binding capture mechanism, antibody- or antibodyfragment-binding capture mechanism, aptamer-binding capture mechanism,and cofactor-binding capture mechanism. In some embodiments of any ofthe systems described herein, the fourth column of the four-column PCCSis a reservoir or a column that is capable of holding eluate from thethree of the four columns of the four-column PCCS containing therecombinant therapeutic protein at low pH for viral inactivation. Insome embodiments of any of the systems described herein, the fourthcolumn of the four-column PCCS is capable of holding the eluatecontaining recombinant therapeutic protein from the three of the fourcolumns of the four-column PCCS at low pH for viral inactivation for aperiod of about 10 minutes to about 1.5 hours.

In some embodiments of any of the systems described herein, the secondMCCS is a second periodic counter current chromatography system (PCCS2).Some embodiments of any of the systems described herein further includea fluid conduit disposed between the first MCCS and the second MCCS.Some embodiments of any of the systems described herein further includean in-line buffer adjustment reservoir in fluid communication with thefluid conduit disposed between the first MCCS and the second MCCS, andconfigured such that buffer contained within the in-line bufferadjustment reservoir is introduced into the fluid present in the fluidconduit disposed between the first MCCS and the second MCCS. Someembodiments of any of the systems described herein further include afilter disposed in the fluid conduit between the first MCCS and thesecond MCCS, and configured such that the filter is capable of removingparticulate matter from the fluid present in the fluid conduit betweenthe first MCCS and the second MCCS.

In some embodiments of any of the systems described herein, the PCCS2includes three chromatography columns and a chromatographic membrane.Some embodiments of any of the methods described herein further includea fluid conduit disposed between the three chromatography columns in thePCCS2 and the chromatographic membrane in the PCCS2. Some embodiments ofany of the systems described herein further include an in-line bufferadjustment reservoir in fluid communication with the fluid conduitdisposed between the three chromatography columns in the PCCS2 and thechromatographic membrane in the PCCS2, and configured such that buffercontained within the in-line buffer adjustment reservoir is introducedinto the fluid present in the fluid conduit disposed between the threechromatography columns in the PCCS2 and the chromatographic membrane inthe PCCS2.

In some embodiments of any of the systems described herein, the threechromatography columns in the PCCS2 are capable of purifying therecombinant therapeutic protein through a cation or anion exchangechromatography. In some embodiments of any of the systems describedherein, the chromatographic membrane in the PCCS2 is a cation exchangechromatographic membrane.

Some embodiments of any of the systems described herein further includea fluid conduit between the chromatographic membrane in the PCCS2 andthe outlet. Some embodiments of any of the systems described hereinfurther include a filter disposed in the fluid conduit between thechromatographic membrane in the PCCS2 and the outlet, and configuredsuch that the filter is capable of removing particulate matter from thefluid present in the fluid conduit between the chromatographic membranein the PCCS2 and the outlet. Some embodiments of any of the systemsdescribed herein further include a pump system that is in fluidcommunication with the inlet. In some embodiments of any of the systemsdescribed herein, the pump system includes a pump that is capable ofpushing the fluid into the inlet. Some embodiments of any of the systemsdescribed herein further include a fluid conduit disposed between thepump and the inlet. Some embodiments of any of the systems describedherein further include a filter disposed in the fluid conduit betweenthe pump and the inlet, and configured such that the filter is capableof removing particulate matter from the fluid present in the fluidconduit between the pump and the inlet. Some embodiments of any of thesystems described herein further include a break tank (e.g., a breaktank described herein) disposed in the fluid conduit between the pumpand the inlet that is configured such that the break tank is in fluidcommunication with the fluid conduit between the pump and the inlet, andis capable of storing any fluid present in the fluid conduit that is notable to enter the inlet.

In some embodiments of any of the systems described herein, the firstMCCS and the second MCCS are disposed on a skid (e.g., a skid includingone or more structures that enable movement). In some embodiments of anyof the systems described herein, the first MCCS is disposed on a firstskid. In some embodiments of any of the systems described herein, thesecond MCCS is disposed on a second skid. In some embodiments of any ofthe systems described herein, the first and second skids each includeone or more structures that enable movement. In some embodiments of anyof the systems described herein, the entire system is disposed on a skid(e.g., a skid that includes one or more structures that enablemovement).

Also provided are biological manufacturing systems that include two ormore subsystems, where the two or more subsystems each include: (i) afirst multi-column chromatography system (MCCS) comprising an inlet; and(ii) a second MCCS comprising an outlet, where the first and secondMCCSs are in fluid communication with each other, and where themanufacturing system is configured such that fluid can be passed intothe inlet, through the first and second MCCS, and exit the manufacturingsystem through the outlet; where the two or more subsystems areconfigured such that they are each in fluid communication with a singlereservoir containing a fluid, and the fluid from the single reservoirpasses into the inlet of the two or more subsystems. In some of theembodiments of any of the systems described herein, each of the two ormore subsystems is each disposed on its own skid. In some of theembodiments of any of the systems described herein, the skid includesone or more structures that enable movement. In some embodiments of anyof the systems described herein, the entire system is disposed on a skid(e.g., a skid that includes one or more structures that enablemovement).

As used herein, the word “a” before a noun represents one or more of theparticular noun. For example, the phrase “a mammalian cell” represents“one or more mammalian cells.”

The term “mammalian cell” means any cell from or derived from any mammal(e.g., a human, a hamster, a mouse, a green monkey, a rat, a pig, a cow,or a rabbit). For example, a mammalian cell can be an immortalized cell.In some embodiments, the mammalian cell is a differentiated cell. Insome embodiments, the mammalian cell is an undifferentiated cell.Non-limiting examples of mammalian cells are described herein.Additional examples of mammalian cells are known in the art.

The term “substantially free” means a composition (e.g., a liquidculture medium) that is at least or about 90% free (e.g., at least orabout 95%, 96%, 97%, 98%, or at least or about 99% free, or about 100%free) of a specified substance (e.g., a mammalian cell).

The term “0.5× volume” means about 50% of the volume. The term “0.6×volume” means about 60% of the volume. Likewise, 0.7×, 0.8×, 0.9×, and1.0× means about 70%, 80%, 90%, or 100% of the volume, respectively.

The term “culturing” or “cell culturing” means the maintenance orproliferation of a mammalian cell under a controlled set of physicalconditions.

The term “culture of mammalian cells” means a liquid culture mediumcontaining a plurality of mammalian cells that is maintained orproliferated under a controlled set of physical conditions.

The term “liquid culture medium” means a fluid that contains sufficientnutrients to allow a cell (e.g., a mammalian cell) to grow orproliferate in vitro. For example, a liquid culture medium can containone or more of: amino acids (e.g., 20 amino acids), a purine (e.g.,hypoxanthine), a pyrimidine (e.g., thymidine), choline, inositol,thiamine, folic acid, biotin, calcium, niacinamide, pyridoxine,riboflavin, thymidine, cyanocobalamin, pyruvate, lipoic acid, magnesium,glucose, sodium, potassium, iron, copper, zinc, and sodium bicarbonate.In some embodiments, a liquid culture medium can contain serum from amammal. In some embodiments, a liquid culture medium does not containserum or another extract from a mammal (a defined liquid culturemedium). In some embodiments, a liquid culture medium can contain tracemetals, a mammalian growth hormone, and/or a mammalian growth factor.Another example of liquid culture medium is minimal medium (e.g., amedium containing only inorganic salts, a carbon source, and water).Non-limiting examples of liquid culture medium are described herein.Additional examples of liquid culture medium are known in the art andare commercially available. A liquid culture medium can contain anydensity of mammalian cells. For example, as used herein, a volume ofliquid culture medium removed from a bioreactor can be substantiallyfree of mammalian cells.

The term “animal-derived component free liquid culture medium” means aliquid culture medium that does not contain any components (e.g.,proteins or serum) derived from a mammal.

The term “serum-free liquid culture medium” means a liquid culturemedium that does not contain a mammalian serum.

The term “serum-containing liquid culture medium” means a liquid culturemedium that contains a mammalian serum.

The term “chemically-defined liquid culture medium” is a term of art andmeans a liquid culture medium in which all of the chemical componentsare known. For example, a chemically-defined liquid culture medium doesnot contain fetal bovine serum, bovine serum albumin, or human serumalbumin, as these preparations typically contain a complex mix ofalbumins and lipids.

The term “protein-free liquid culture medium” means a liquid culturemedium that does not contain any protein (e.g., any detectable protein).

The term “agitation” means stirring or otherwise moving a portion ofliquid culture medium in a bioreactor. This is performed in order to,e.g., increase the dissolved O₂ concentration in the liquid culturemedium in a bioreactor. Agitation can be performed using any art knownmethod, e.g., an instrument or propellor. Exemplary devices and methodsthat can be used to perform agitation of a portion of the liquid culturemedium in a bioreactor are known in the art.

The term “therapeutic protein drug substance” means a recombinantprotein (e.g., an immunoglobulin, protein fragment, engineered protein,or enzyme) that has been sufficiently purified or isolated fromcontaminating proteins, lipids, and nucleic acids (e.g., contaminatingproteins, lipids, and nucleic acids present in a liquid culture mediumor from a host cell (e.g., from a mammalian, yeast, or bacterial hostcell)) and biological contaminants (e.g., viral and bacterialcontaminants), and can be formulated into a pharmaceutical agent withoutany further substantial purification and/or decontamination step.

The term “integrated process” means a process which is performed usingstructural elements that function cooperatively to achieve a specificresult (e.g., the generation of a therapeutic protein drug substancefrom a liquid culture medium).

The term “continuous process” means a process which continuously feedsfluid through at least a part of the system. For example, in any of theexemplary continuous biological manufacturing systems described herein,a liquid culture medium containing a recombinant therapeutic protein iscontinuously fed into the system while it is in operation and atherapeutic protein drug substance is fed out of the system. In anotherexample, a continuous process is a process which continuously feeds aliquid culture medium containing a recombinant therapeutic protein froma bioreactor through a first MCCS. Another example of a continuousprocess is a process which continuously feeds a liquid culture mediumcontaining a recombinant therapeutic protein from a bioreactor through afirst and second MCCS. Additional examples include a process whichcontinuously feeds a liquid culture medium containing a recombinanttherapeutic protein through a first MCCS, a process that continuouslyfeeds a liquid culture medium containing a recombinant therapeuticprotein through a first and second MCCS, or a process that continuouslyfeeds a fluid containing a recombinant therapeutic protein through asecond MCCS.

The term “immunoglobulin” means a polypeptide containing an amino acidsequence of at least 15 amino acids (e.g., at least 20, 30, 40, 50, 60,70, 80, 90, or 100 amino acids) of an immunoglobulin protein (e.g., avariable domain sequence, a framework sequence, or a constant domainsequence). The immunoglobulin may, for example, include at least 15amino acids of a light chain immunoglobulin, e.g., at least 15 aminoacids of a heavy chain immunoglobulin. The immunoglobulin may be anisolated antibody (e.g., an IgG, IgE, IgD, IgA, or IgM). Theimmunoglobulin may be a subclass of IgG (e.g., IgG1, IgG2, IgG3, orIgG4). The immunoglobulin may be an antibody fragment, e.g., a Fabfragment, a F(ab′)₂ fragment, or an a scFv fragment. The immunoglobulinmay also be a bi-specific antibody or a tri-specific antibody, or adimer, trimer, or multimer antibody, or a diabody, an Affibody®, or aNanobody®. The immunoglobulin can also be an engineered proteincontaining at least one immunoglobulin domain (e.g., a fusion protein).Non-limiting examples of immunoglobulins are described herein andadditional examples of immunoglobulins are known in the art.

The term “protein fragment” or “polypeptide fragment” means a portion ofa polypeptide sequence that is at least or about 4 amino acids, at leastor about 5 amino acids, at least or about 6 amino acids, at least orabout 7 amino acids, at least or about 8 amino acids, at least or about9 amino acids, at least or about 10 amino acids, at least or about 11amino acids, at least or about 12 amino acids, at least or about 13amino acids, at least or about 14 amino acids, at least or about 15amino acids, at least or about 16 amino acids, at least or about 17amino acids, at least or about 18 amino acids, at least or about 19amino acids, or at least or about 20 amino acids in length, or more than20 amino acids in length. A recombinant protein fragment can be producedusing any of the processes described herein.

The term “engineered protein” means a polypeptide that is not naturallyencoded by an endogenous nucleic acid present within an organism (e.g.,a mammal). Examples of engineered proteins include enzymes (e.g., withone or more amino acid substitutions, deletions, insertions, oradditions that result in an increase in stability and/or catalyticactivity of the engineered enzyme), fusion proteins, antibodies (e.g.,divalent antibodies, trivalent antibodies, or a diabody), andantigen-binding proteins that contain at least one recombinantscaffolding sequence.

The term “multi-column chromatography system” or “MCCS” means a systemof a total of two or more interconnected or switching chromatographycolumns and/or chromatographic membranes. A non-limiting example of amulti-column chromatography system is a periodic counter currentchromatography system (PCC) containing a total of two or moreinterconnected or switching chromatography columns and/orchromatographic membranes. Additional examples of multi-columnchromatography systems are described herein and are known in the art.

The term “capturing” means a step performed to partially purify orisolate (e.g., at least or about 5%, e.g., at least or about 10%, 15%,20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, orat least or about 95% pure by weight), concentrate, and stabilize arecombinant therapeutic protein from one or more other componentspresent in a liquid culture medium or a diluted liquid culture medium(e.g., culture medium proteins or one or more other components (e.g.,DNA, RNA, or other proteins) present in or secreted from a mammaliancell). Typically, capturing is performed using a resin that binds arecombinant therapeutic protein (e.g., through the use of affinitychromatography). Non-limiting methods for capturing a recombinanttherapeutic protein from a liquid culture medium or diluted liquidculture medium are described herein and others are known in the art. Arecombinant therapeutic protein can be captured from a liquid culturemedium using at least one chromatography column and/or chromatographicmembrane (e.g., any of the chromatography columns and/or chromatographicmembranes described herein).

The term “purifying” means a step performed to isolate a recombinanttherapeutic protein from one or more other impurities (e.g., bulkimpurities) or components present in a fluid containing a recombinanttherapeutic protein (e.g., liquid culture medium proteins or one or moreother components (e.g., DNA, RNA, other proteins, endotoxins, viruses,etc.) present in or secreted from a mammalian cell). For example,purifying can be performed during or after an initial capturing step.Purification can be performed using a resin, membrane, or any othersolid support that binds either a recombinant therapeutic protein orcontaminants (e.g., through the use of affinity chromatography,hydrophobic interaction chromatography, anion or cation exchangechromatography, or molecular sieve chromatography). A recombinanttherapeutic protein can be purified from a fluid containing therecombinant therapeutic protein using at least one chromatography columnand/or chromatographic membrane (e.g., any of the chromatography columnsor chromatographic membranes described herein).

The term “polishing” is a term of art and means a step performed toremove remaining trace or small amounts of contaminants or impuritiesfrom a fluid containing a recombinant therapeutic protein that is closeto a final desired purity. For example, polishing can be performed bypassing a fluid containing the recombinant therapeutic protein through achromatographic column(s) or membrane absorber(s) that selectively bindsto either the target recombinant therapeutic protein or small amounts ofcontaminants or impurities present in a fluid containing a recombinanttherapeutic protein. In such an example, the eluate/filtrate of thechromatographic column(s) or membrane absorber(s) contains therecombinant therapeutic protein.

The term “eluate/filtrate” is a term of art and means a fluid that isemitted from a chromatography column or chromatographic membrane thatcontains a detectable amount of a recombinant therapeutic protein.

The term “filtering” means the removal of at least part of (e.g., atleast 80%, 90%, 95%, 96%, 97%, 98%, or 99%) undesired biologicalcontaminants (e.g., a mammalian cell, bacteria, yeast cells, viruses, ormycobacteria) and/or particulate matter (e.g., precipitated proteins)from a liquid (e.g., a liquid culture medium or fluid present in any ofthe systems or processes described herein).

The term “secreted protein” or “secreted recombinant protein” means aprotein (e.g., a recombinant protein) that originally contained at leastone secretion signal sequence when it is translated within a mammaliancell, and through, at least in part, enzymatic cleavage of the secretionsignal sequence in the mammalian cell, is secreted at least partiallyinto the extracellular space (e.g., a liquid culture medium). Skilledpractitioners will appreciate that a “secreted” protein need notdissociate entirely from the cell to be considered a secreted protein.

The term “perfusion bioreactor” means a bioreactor containing aplurality of cells (e.g., mammalian cells) in a first liquid culturemedium, wherein the culturing of the cells present in the bioreactorincludes periodic or continuous removal of the first liquid culturemedium and at the same time or shortly thereafter adding substantiallythe same volume of a second liquid culture medium to the bioreactor. Insome examples, there is an incremental change (e.g., increase ordecrease) in the volume of the first liquid culture medium removed andadded over incremental periods (e.g., an about 24-hour period, a periodof between about 1 minute and about 24-hours, or a period of greaterthan 24 hours) during the culturing period (e.g., the culture mediumrefeed rate on a daily basis). The fraction of media removed andreplaced each day can vary depending on the particular cells beingcultured, the initial seeding density, and the cell density at aparticular time. “RV” or “reactor volume” means the volume of theculture medium present at the beginning of the culturing process (e.g.,the total volume of the culture medium present after seeding).

The term “fed-batch bioreactor” is a term of art and means a bioreactorcontaining a plurality of cells (e.g., mammalian cells) in a firstliquid culture medium, wherein the culturing of the cells present in thebioreactor includes the periodic or continuous addition of a secondliquid culture medium to the first liquid culture medium withoutsubstantial or significant removal of the first liquid culture medium orsecond liquid culture medium from the cell culture. The second liquidculture medium can be the same as the first liquid culture medium. Insome examples of fed-batch culture, the second liquid culture medium isa concentrated form of the first liquid culture medium. In some examplesof fed-batch culture, the second liquid culture medium is added as a drypowder.

The term “clarified liquid culture medium” means a liquid culture mediumobtained from a bacterial or yeast cell culture that is substantiallyfree (e.g., at least 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 99% free) ofbacteria or yeast cells.

The term “unit operation” is a term of art and means a functional stepthat can be performed in a process of manufacturing a therapeuticprotein drug substance from a liquid culture medium. For example, a unitof operation can be filtering (e.g., removal of contaminant bacteria,yeast viruses, or mycobacteria, and/or particular matter from a fluidcontaining a recombinant therapeutic protein), capturing, epitope tagremoval, purifying, holding or storing, polishing, viral inactivating,adjusting the ionic concentration and/or pH of a fluid containing therecombinant therapeutic protein, and removing unwanted salts.

“Specific productivity rate” or “SPR” is a term of art and as usedherein refers to the mass or enzymatic activity of a recombinanttherapeutic protein produced per mammalian cell per day. The SPR for arecombinant therapeutic antibody is usually measured as mass/cell/day.The SPR for a recombinant therapeutic enzyme is usually measured asunits/cell/day or (units/mass)/cell/day.

“Volume productivity rate” or “VPR” is a term of art and as used hereinrefers to the mass or enzymatic activity of recombinant therapeuticprotein produced per volume of culture (e.g., per L of bioreactor,vessel, or tube volume) per day. The VPR for a recombinant therapeuticantibody is usually measured as mass/L/day. The VPR for a recombinanttherapeutic enzyme is usually measured as units/L/day or mass/L/day.

“Skid” is a term of art and as used herein refers to a three-dimensionalsolid structure that can act as a platform or support for a systemdescribed herein. A skid can, if it comprises one or more structuresthat enable movement (e.g., wheels, rollers, or the like), confermobility on the system or a portion thereof. Non-limiting examples ofskids are described herein. Additional examples of skids are known inthe art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing an exemplary integrated systemthat can be used to continuously produce a recombinant protein drugsubstance.

FIG. 2 is a diagram of the single PCC system connected to a perfusioncell culture bioreactor that can be used to capture a recombinanttherapeutic protein present in the cell culture medium obtained from thebioreactor.

FIG. 3 is a diagram of a PCC system cycle containing threechromatography columns. At the beginning of a cycle, the feed solutionis loaded onto column 1, and the flow through goes to waste untilproduct breakthrough occurs (step 1). At this point, the flow throughfrom column 1 is directed to column 2 to capture the unbound recombinanttherapeutic protein from column 1 (step 2). Once column 1 is fullyloaded, the feed is now directed loaded onto column 2, while column 1 iswashed, eluted, regenerated, and re-equilibrated for the next cycle(steps 3 and 4). Column 2 now goes through steps 3-5, which areidentical to steps 1-3 for column 1. Finally, column 3 goes throughsteps 5-6, the same as columns 1 and 2. Once all three columns havecompleted these steps, the cycle restarts with column 1.

FIG. 4 is a schematic diagram demonstrating the principle of columnswitching based on ΔUV. T1 designates the time when the ΔUV has reacheda pre-determined threshold level. Once this threshold level is reached,the flow through from column 1 is directed onto column 2 rather than tothe waste. T2 designates the time when the column has been saturatedwith product. The ΔUV value for both T1 and T2 are process specific.

FIG. 5A is a graph of the cell density profile over time in thebioreactor culture producing the recombinant therapeutic antibody. Theaverage cell density in the bioreactor culture was 50-60×10⁶ cells/mL.

FIG. 5B is a graph of the volumetric productivity rate over time of thebioreactor culture producing the recombinant therapeutic antibody.

FIG. 6 is a set of graphs showing the percent recovery, percentaggregation, potency (as compared to control), residual protein Aconcentration (ng/mg), and host cell protein concentration (ng/mg) inthe recombinant therapeutic protein eluted from the single PCC systemover a total of 30 days, 38 single PCC system cycles, and 110 columnoperations. The variability associated with the three ELISA assays(recovery, residual protein A, and host cell protein) was ±20%. All ofthe data shown are within the assay variability.

FIG. 7A is a graph of the cell density profile over time in thebioreactor culture producing the recombinant therapeutic human enzyme.The average cell density in the bioreactor culture after day 19 was50-60×10⁶ cells/mL.

FIG. 7B is a graph of the volumetric productivity rate over time of thebioreactor culture producing the recombinant therapeutic human enzyme.The two low titer values determined around day 48-49 are believed to bedue to assay variability as there were no changes in cell density,perfusion rate, or metabolism over this period.

FIG. 8 is a set of graphs showing the percent recovery, specificactivity (units/mg), percent aggregation, potency (as compared tocontrol), Critical Quality Attribute (CQA) #1, CQA #2, CQA #3, and CQA#4 in the recombinant therapeutic human enzyme eluted from the singlePCC system over a total of 9 days, 41 single PCC system cycles, and 164column operations. CQAs #3 and #4 have limited data points, butrepresent the entire operational duration. The standard deviation of theCQAs is in the range of 1-9%.

FIG. 9 is a schematic showing the two-PCCS manufacturing systemconnected with a perfusion culture bioreactor that results in thecontinuous manufacture of a therapeutic protein drug substance.

FIG. 10 is a graph of the viable cell density profile over time in thebioreactor culture producing the recombinant therapeutic monoclonalantibody. The average cell density in the bioreactor culture was50-60×10⁶ cells/mL.

FIG. 11 is a graph of the volumetric productivity rate over time of thebioreactor culture producing the recombinant therapeutic monoclonalantibody.

FIG. 12 is a graph of the real-time UV profile of the Protein A capturecolumns over a limited time window. Every third peak of every threepeaks represents the column 1 outlet UV absorbance, every second peak ofevery three peaks represents the column 2 UV outlet absorbance, everyfirst peak of every three peaks represents the column 3 UV absorbance.

FIG. 13 is a graph of the real-time UV profile of the Capto-S columnsover a limited time window. The dashed black line represents the feed UVabsorbance, every third peak of every three peaks represents the column1 outlet UV absorbance, every second peak of every three peaksrepresents the column 2 UV outlet absorbance, and every first peak ofevery three peaks represents the column 3 UV absorbance.

FIG. 14 is a graph of the real-time UV profile of the SartobindQ-membrane over a limited time window.

FIG. 15 is a sodium dodecyl sulfate polyacrylamide electrophoresis gelof a reference amount of the recombinant monoclonal antibody (lane 1),and the eluate of the two-PCC system obtained at days 1, 10, 15, 20, 25,30, or 31 of production (lanes 3-9, respectively).

FIG. 16 is a set of graphs showing the protein concentration (mg/mL),percent aggregation, potency (as compared to control), residual proteinA concentration (ng/mg), and host cell protein concentration (ng/mg) inthe product eluted from the two-PCC system over a total of 31 days and25 dual system batches. The variability associated with the aggregationand residual protein A measurements was due to the unoptimized Qmembrane operation. The trends were, however, comparable between thebatch and continuous operation. All the results above are within theassay variability.

DETAILED DESCRIPTION

Provided herein are integrated and fully continuous processes formanufacturing a therapeutic protein drug substance. These processesinclude, e.g., providing a liquid culture medium containing arecombinant therapeutic protein that is substantially free of cells,then feeding the liquid culture medium into a first multi-columnchromatography system (MCCS1). The next step involves capturing therecombinant therapeutic protein in the liquid culture medium using theMCCS1. The next steps involve continuously feeding the eluate of theMCCS1 containing the recombinant therapeutic protein into a secondmulti-column chromatography system (MCCS2), and purifying and polishingthe protein using the MCCS2. The resulting eluate from the MCCS2 isconsidered a therapeutic protein drug substance. The processes areintegrated and can run continuously from the liquid culture medium tothe eluate from the MCCS2 that is the therapeutic protein drugsubstance. Also provided herein are exemplary biological manufacturingsystems that can be used to perform these methods.

Biological Manufacturing Systems

The present specification provides exemplary biological manufacturingsystems useful for performing the processes described herein. Forexample, useful systems can include a first multi-column chromatographysystem (MCCS) that includes an inlet and a second MCCS that includes anoutlet. In these systems, the first and second MCCSs are in fluidcommunication with each other. The systems are also configured such thatfluid can be passed into the inlet, through the first and second MCCSs,and exit the manufacturing system through the outlet.

The systems described herein provide for the continuous andtime-efficient production of a therapeutic drug substance from a liquidculture medium. For example, the elapsed time between feeding a fluid(e.g., a liquid culture medium) containing a therapeutic protein intothe first MCCS and eluting a therapeutic protein drug substance(containing the therapeutic protein) from the outlet of the second MCCScan be, e.g., between about 4 hours and about 48 hours, inclusive, e.g.,between about 4 hours and about 40 hours, between about 4 hours andabout 35 hours, between about 4 hours and about 30 hours, between about4 hours and about 28 hours, between about 4 hours and about 26 hours,between about 4 hours and about 24 hours, between about 4 hours andabout 22 hours, between about 4 hours and about 20 hours, between about4 hours and about 18 hours, between about 4 hours and about 16 hours,between about 4 hours and about 14 hours, between about 4 hours andabout 12 hours, between about 6 hours and about 12 hours, between about8 hours and about 12 hours, between about 6 hours and about 20 hours,between about 6 hours and about 18 hours, between about 6 hours andabout 14 hours, between about 8 hours and about 16 hours, between about8 hours and about 14 hours, between about 8 hours and about 12 hours,between about 10 hours and 20 hours, between about 10 hours and 18hours, between about 10 hours and 16 hours, between about 10 hours and14 hours, between about 12 hours and about 14 hours, between about 10hours and about 40 hours, between about 10 hours and about 35 hours,between about 10 hours and about 30 hours, between about 10 hours andabout 25 hours, between about 15 hours and about 40 hours, between about15 hours and about 35 hours, between about 15 hours and about 30 hours,between about 20 hours and about 40 hours, between about 20 hours andabout 35 hours, or between about 20 hours and about 30 hours, inclusive.In other examples, the elapsed time between feeding the fluid (e.g., aliquid culture medium) containing a therapeutic protein into the firstMCCS and eluting a therapeutic protein drug substance (containing thetherapeutic protein) from the outlet of the second MCCS is, e.g.,greater than about 4 hours and is less than about 40 hours, inclusive,e.g., greater than about 4 hours and less than about 39 hours, about 38hours, about 37 hours, about 36 hours, about 35 hours, about 34 hours,about 33 hours, about 32 hours, about 31 hours, about 30 hours, about 29hours, about 28 hours, about 27 hours, about 26 hours, about 25 hours,about 24 hours, about 23 hours, about 22 hours, about 21 hours, about 20hours, about 19 hours, about 18 hours, about 17 hours, about 16 hours,about 15 hours, about 14 hours, about 13 hours, about 12 hours, about 11hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours,about 6 hours, about 5 hours, or about 4.5 hours, inclusive.

Some exemplary systems do not contain a break tank. In others, thesystem can contain a maximum of 1, 2, 3, 4, or 5 break tank(s) in theentire system. Any of the systems described herein can contain, e.g., amaximum of 1, 2, 3, 4, or 5 break tank(s) in the entire system, whereeach break tank only holds a therapeutic protein for a total time periodof, e.g., between about 5 minutes and about 6 hours, inclusive, e.g.,between about 5 minutes and about 5 hours, about 4 hours, about 3 hours,about 2 hours, about 1 hour, or about 30 minutes, inclusive. Breaktank(s) described herein can have a capacity that is between 1 mL andabout 300 mL, inclusive, between about 1 mL and about 280 mL, about 260mL, about 240 mL, about 220 mL, about 200 mL, about 180 mL, about 160mL, about 140 mL, about 120 mL, about 100 mL, about 80 mL, about 60 mL,about 40 mL, about 20 mL, or about 10 mL, inclusive. Any break tank(s)disposed in the system such that fluid enters the break tank(s) prior toentering MCCS1 can have a capacity that is between 1 mL and about 100%,inclusive, e.g., between about 1 mL and about 90%, about 80%, about 70%,about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, orabout 5%, inclusive, of the loading volume of the first column of theMCCS1. Any break tanks(s) disposed in the system such that fluid entersthe break tank(s) prior to entering the MCCS2 (and after exiting theMCCS1) can have a capacity that is, e.g., between 1 mL and about 100%,inclusive, e.g., between about 1 mL and about 90%, about 80%, about 70%,about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, orabout 5%, inclusive, of the loading volume of the first column of theMCCS2.

Exemplary System

A non-limiting example of a system 1 useful in the present invention isprovided in FIG. 1. System 1 includes a first MCCS, i.e., a four-columnPCCS 2, where three of the four columns 3, 4, and 5 in four-column PCCS2 perform the unit operation of capturing the recombinant therapeuticprotein from a fluid containing the recombinant therapeutic protein(e.g., liquid culture medium that is substantially free of mammaliancells), and one of the columns 6 in PCCS 2 performs the unit operationof inactiving viruses present in the eluate from columns 3, 4, and 5 inPCCS 2 containing the recombinant therapeutic protein. Columns 3, 4, and5 can contain a resin that utilizes a protein A-binding capturemechanism. Column 6 is capable of holding a fluid at a pH of about 3.75for about 1 hour. PCCS 1 also has an inlet 7. Inlet 7 can be, e.g., anoriface that accepts entry of a fluid into PCCS 1.

System 1 also includes a second MCCS that is a PPCS 8 that includesthree chromatography columns 9, 10, and 11 and one chromatographicmembrane 12. Columns 9, 10, and 11 in PCCS 8 can contain a cationicexchange resin. Chromatographic membrane 12 in PCCS 8 can contain acationic exchange resin. PCCS 8 also has a fluid conduit 13 disposedbetween columns 9, 10, and 11 in PCCS 8 and chromatographic membrane 12in PCCS 8. PCCS 8 also has an in-line buffer adjustment reservoir 14that is in fluid communication with fluid conduit 13, and is configuredsuch that buffer contained within in-line buffer adjustment reservoir 14is introduced into the fluid present in fluid conduit 13. PCCS 8 alsoincludes an outlet 15. Outlet 15 can be, e.g., an orifice that allowsexit of the fluid from PCCS 8.

System 1 may further include a fluid conduit 16 disposed between PCC1 2and PCC2 8. System 1 may also include an in-line buffer adjustmentreservoir 17 in fluid communication with fluid conduit 16 configuredsuch that the buffer contained within in-line buffer adjustmentreservoir 17 can be introduced into the fluid present in fluid conduit16. System 1 may also include a filter 18 disposed in fluid conduit 16to filter the fluid present in fluid conduit 16. System 1 may alsoinclude a second first break tank 19 disposed in fluid conduit 16 andconfigured to hold any fluid in fluid conduit 11 that cannot be readilyfed into PCCS 8.

System 1 may further include a pump system 20 that is in fluidcommunication with inlet 7. Pump system 20 may include a pump 21 forpushing fluid into inlet 7. System 1 may also include a fluid conduit 22disposed between pump 21 and inlet 7. System 1 may also include a filter23 disposed in fluid conduit 22 to filter the fluid (e.g., liquidculture medium) present in fluid conduit 22. System 1 may also include abreak tank 24 disposed in fluid conduit 22 configured such that breaktank 24 is in fluid communication with fluid conduit 22 and is capableof storing any fluid present in fluid conduit 22 that is not able toenter inlet 7.

System 1 may also include a bioreactor 25 and a fluid conduit 26disposed between bioreactor 25 and pump 21. A filtration system 27 maybe disposed in fluid conduit 26 to filter (e.g., remove cells) from aliquid culture medium present in fluid conduit 26.

Additional Exemplary System Structures and Features

The first MCCS includes an inlet through which fluid (e.g., a liquidculture medium that is substantially free of cells) can be passed intothe first MCCS. The inlet can be any structure known in the art for suchpurposes. It can include, e.g., a threading, ribbing, or a seal thatallows for a fluid conduit to be inserted, such that after insertion ofthe fluid conduit into the inlet, fluid will enter the first MCCSthrough the inlet without significant seepage of fluid out of the inlet.Non-limiting inlets that can be used in the present systems are knownand would be understood by those in the art.

First MCCS

The first MCCS includes at least two chromatography columns, at leasttwo chromatographic membranes, or at least one chromatography column andat least one chromatographic membrane, and an inlet. For example, thefirst MCCS can include a total of four chromatography columns, or athree chromatography columns and one chromatographic membrane, or any ofthe other exemplary MCCSs described herein, or have one or more of anyof the exemplary features of an MCCS (in any combination) describedherein. The chromatography column(s) and/or the chromatographicmembrane(s) present in the first MCCS can have one or more of any of theexemplary shapes, sizes, volumes (bed volumes), and/or unit operation(s)described herein.

The chromatography column(s) and/or the chromatographic membrane(s)present in the first MCCS can contain one or more of any of theexemplary resins described herein or known in the art. For example, theresin contained in one or more of the chromatography column(s) and/orchromatographic membrane(s) present in the first MCCS can be a resinthat utilizes a capture mechanism (e.g., protein A-binding capturemechanism, protein G-binding capture mechanism, antibody- or antibodyfragment-binding capture mechanism, substrate-binding capture mechanism,cofactor-binding capture mechanism, an aptamer-binding capturemechanism, and/or a tag-binding capture mechanism). The resin containedin one or more of the chromatography column(s) and/or chromatographicmembrane(s) of the first MCCS can be a cation exchange resin, an anionexchange resin, a molecular sieve resin, or a hydrophobic interactionresin, or any combination thereof. Additional examples of resins thatcan be used to purify a recombinant therapeutic protein are known in theart, and can be contained in one or more of the chromatography column(s)and/or chromatographic membrane(s) present in the first MCCS. Thechromatography column(s) and/or chromatography membranes present in thefirst MCCS can contain the same and/or different resins (e.g., any ofthe resins described herein or known in the art for use in recombinantprotein purification).

The two or more chromatography column(s) and/or chromatographic resin(s)present in the first MCCS can perform one or more unit operations (e.g.,capturing a recombinant therapeutic protein, purifying a recombinanttherapeutic protein, polishing a recombinant therapeutic protein,inactivating viruses, adjusting the ionic concentration and/or pH of afluid containing the recombinant therapeutic protein, or filtering afluid containing a recombinant therapeutic protein). In non-limitingexamples, the first MCCS can perform the unit operations of capturing arecombinant therapeutic protein from a fluid (e.g., a liquid culturemedium) and inactivating viruses present in the fluid containing therecombinant therapeutic protein. The first MCCS can perform anycombinations of two of more unit operations described herein or known inthe art.

The chromatography column(s) and/or chromatographic membrane(s) presentin the first MCCS can be connected or moved with respect to each otherby a switching mechanism (e.g., a column-switching mechanism). The firstMCCS can also include one or more (e.g., two, three, four, or five)pumps (e.g., automated, e.g., automated peristaltic pumps). Thecolumn-switching events can be triggered by the detection of a level ofrecombinant therapeutic protein detected by UV absorbance correspondingto a certain level of recombinant therapeutic protein in the fluidpassing through the first MCCS (e.g., the input into and/or eluate fromone or more of the chromatography column(s) and/or chromatographicmembranes in the first MCCS), a specific volume of liquid (e.g.,buffer), or specific time elapsed. Column switching generally means amechanism by which at least two different chromatography columns and/orchromatographic membranes in an MCCS (e.g., two or more differentchromatography columns and/or chromatographic membranes present in anMCCS (e.g., the first or second MCCS)) are allowed to pass through adifferent step (e.g., equilibration, loading, eluting, or washing) atsubstantially the same time during at least part of the process.

The first MCCS can be a Periodic Counter-Current Chromatography system(PCCS). For example, the PCCS that is the first MCCS can include fourchromatography columns, where the first three columns perform the unitoperation of capturing a recombinant therapeutic protein from a fluid(e.g., a liquid culture medium), and the fourth column of the PCCSperforms the unit operation of inactivating viruses in the fluidcontaining the recombinant therapeutic protein. A PCCS that is the firstMCCS can utilize a column-switching mechanism. The PCC system canutilize a modified ÄKTA system (GE Healthcare, Piscataway, N.J.) capableof running up to, e.g., four, five, six, seven, or eight columns, ormore.

The first MCCS can be equipped with: one or more (e.g., two, three,four, five, six, seven, eight, nine, or ten) UV monitors, one or more(e.g., two, three, four, five, six, seven, eight, nine, or ten) valves,one or more (e.g., two, three, four, five, six, seven, eight, nine, orten) pH meters, and/or one or more (e.g., two, three, four, five, six,seven, eight, nine, or ten) conductivity meters. The first MCCS can alsobe equipped with an operating system that utilizes software (e.g.,Unicorn-based software, GE Healthcare, Piscataway, N.J.) for sensingwhen a column-switching should occur (e.g., based upon UV absorbance,volume of liquid, or time elapsed) and affecting (triggering) thecolumn-switching events. In the examples where MCCS includes one or moreUV detectors, the UV detectors can be placed optionally at the inlet ofone or more (e.g., two, three, four, five, six, seven, eight, nine, orten) of the chromatography column(s) and/or chromatographic membrane(s)in the first MCCS, and/or at the outlet of one or more of thechromatography column(s) and/or chromatography membrane(s) in the firstMCCS.

The first MCCS can further include one or more (e.g., two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one,twenty-two, twenty-three, or twenty-four) in-line buffer adjustmentreservoir(s) and/or a buffer reservoir(s). In other examples, the firstMCCS can include one or more (e.g., two, three, four, five, or six)break tanks that can hold fluid that cannot readily pass into one ormore of the chromatography columns and/or chromatographic membranes inthe first MCCS. The systems described herein can contain one or morebreak tanks (e.g., a break tank described herein) in the first and/orsecond MCCS. Other examples of the systems described herein do notinclude a break tank in the first MCCS or the second MCCS, or do notinclude a break tank in the entire system. Other examples of the systemsdescribed herein contain a maximum of one, two, three, four, or fivebreak tank(s) (e.g., any break tank(s) described herein) in the entiresystem.

Second MCCS

The second MCCS includes at least two chromatography columns, at leasttwo chromatographic membranes, or at least one chromatography column(s)and at least one chromatographic membrane(s), and an outlet. Forexample, the second MCCS can include a total of four chromatographycolumns, three chromatography columns and one chromatographic membrane,or any of the other exemplary MCCSs described herein, or can have one ormore of any of the exemplary features of an MCCS (in any combination)described herein. The chromatography column(s) and/or thechromatographic membrane(s) present in the second MCCS can have one ormore of: any of the shapes, sizes, volumes (bed volumes), and/or unitoperations described herein. The chromatography column(s) and/or thechromatographic membrane(s) can contain any of the exemplary resinsdescribed herein or known in the art. For example, the resin containedin one or more of the chromatography column(s) and/or chromatographicmembrane(s) present in the second MCCS can be a resin that utilizes acapture mechanism (e.g., protein A-binding capture mechanism, proteinG-binding capture mechanism, antibody- or antibody fragment-bindingcapture mechanism, substrate-binding capture mechanism, cofactor-bindingcapture mechanism, tag-binding capture mechanism, and/or aptamer-bindingcapture mechanism). Useful resins include, e.g., a cation exchangeresin, an anion exchange resin, a molecular sieve resin, and ahydrophobic interaction resin. Additional examples of resins are knownin the art. The chromatography column(s) and/or chromatography membranespresent in the second MCCS can contain the same and/or different resins(e.g., any of the resins described herein or known in the art for use inrecombinant protein purification).

The chromatography column(s) and/or chromatographic membrane(s) presentin the second MCCS can perform one or more unit operations (e.g., any ofthe unit operations described herein or any combination of the unitoperations described herein). In non-limiting examples, the second MCCScan perform the unit operations of purifying a recombinant therapeuticprotein from a fluid and polishing the recombinant therapeutic proteinpresent in the fluid containing the recombinant therapeutic protein. Inother non-limiting examples, the second MCCS can perform the unitoperations of purifying a recombinant therapeutic protein present in afluid, polishing a recombinant therapeutic protein present in a fluid,and filtering a fluid containing a recombinant therapeutic protein. Inanother example, the second MCCS can perform the unit operations ofpurifying a recombinant therapeutic protein present in a fluid,polishing a recombinant therapeutic protein present in a fluid,filtering a fluid containing a recombinant therapeutic protein, andadjusting the ionic concentration and/or pH of a fluid containing arecombinant therapeutic protein. The second MCCS can perform anycombination of two of more unit operations described herein or known inthe art.

The chromatography column(s) and/or chromatographic membrane(s) presentin the second MCCS can be connected or moved with respect to each otherby a switching mechanism (e.g., a column-switching mechanism). Thesecond MCCS can also include one or more (e.g., two, three, four, orfive) pumps (e.g., automated, e.g., automated peristaltic pumps). Thecolumn-switching events can be triggered by the detection of a level ofrecombinant therapeutic protein detected by UV absorbance correspondingto a certain level of recombinant therapeutic protein in the fluidpassing through the second MCCS (e.g., the input into and/or eluate fromone or more of the chromatography column(s) and/or chromatographicmembranes in the second MCCS), a specific volume of liquid (e.g.,buffer), or specific time elapsed.

The second MCCS be a Periodic Counter-Current Chromatography system(PCCS). For example, the PCCS that is the second MCCS can contain threecolumns that perform the unit operation of purifying a recombinanttherapeutic protein from a fluid, and a chromatographic membrane thatperforms the unit operation of polishing a recombinant therapeuticprotein present in a fluid. For example, the three columns that performthe unit operation of purifying a recombinant therapeutic protein from afluid can contain, e.g., a cationic exchange resin, and thechromatographic membrane that performs the unit operation of polishingcan contain a cationic exchange resin. A PCCS that is the second MCCScan utilize a column-switching mechanism. The PCC system can utilize amodified ÄKTA system (GE Healthcare, Piscataway, N.J.) capable ofrunning up to, e.g., four, five, six, seven, or eight columns, or more.

The second MCCS can be equipped with: one or more (e.g., two, three,four, five, six, seven, eight, nine, or ten) UV monitors, one or more(e.g., two, three, four, five, six, seven, eight, nine, or ten) valves,one or more (e.g., two, three, four, five, six, seven, eight, nine, orten) pH meters, and/or one or more (e.g., two, three, four, five, six,seven, eight, nine, or ten) conductivity meters. The second MCCS canalso be equipped with an operating system that utilizes software (e.g.,Unicorn-based software, GE Healthcare, Piscataway, N.J.) for sensingwhen a column-switching event should occur (e.g., based upon UVabsorbance, volume of liquid, or time elapsed) and affecting thecolumn-switching events. In the examples where the second MCCS includesone or more UV detectors, the UV detectors can be placed optionally atthe inlet of one or more (e.g., two, three, four, five, six, seven,eight, nine, or ten) of the chromatography column(s) and/orchromatographic membrane(s) in the second MCCS, and/or at the outlet ofone or more of the chromatography column(s) and/or chromatographymembrane(s) in the second MCCS.

The second MCCS can further include one or more (e.g., two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one,twenty-two, twenty-three, or twenty-four) in-line buffer adjustmentreservoir(s) and/or a buffer reservoir(s). In other examples, the secondMCCS can include one or more (e.g., two, three, four, five, or six)break tanks (e.g., any of the break tanks described herein) that canhold fluid that cannot readily pass into one or more of thechromatography columns and/or chromatographic membranes in the secondMCCS.

The second MCCS includes an outlet through which the therapeutic proteindrug substance can exit the system. The outlet can include, e.g., athreading, ribbing, or a seal that allows for a fluid conduit to beinserted or a vial designed to contain or store the therapeutic proteindrug substance. An outlet can contain a surface that can be used to seala sterile vial or other such storage container onto the outlet in orderto allow the recombinant protein drug product to flow directly into thesterile vial or storage container. Non-limiting outlets that can be usedin the present systems are known and would be understood by those in theart.

The systems described herein can also include a fluid conduit that isdisposed between the first MCCS and the second MCCS. Any of the fluidconduits described herein can be, e.g., a tube that is made of, e.g.,polyethylene, polycarbonate, or plastic. The fluid conduit disposedbetween the first MCCS and the second MCCS can further include one ofmore of the following in any combination: one or more in-line bufferadjustment reservoirs that are in fluid communication with the fluidconduit and are positioned such that the buffer stored within thein-line buffer adjustment reservoir(s) is added to the fluid present inthe fluid conduit; a break tank (e.g., any of the break tank(s)described herein) that is in fluid communication with the fluid conduitand is positioned such that it can hold any excess fluid present in thefluid conduit that is unable to readily feed into the second MCCS; andone or more filters that are disposed in the fluid conduit such thatthey are capable of filtering (e.g., removing bacteria) the fluidpresent in the fluid conduit. Any of the in-line buffer adjustmentreservoirs can contain, e.g., a volume of between about 0.5 L to 50 L ofbuffer (e.g., at a temperature at or below 25° C., 15° C., or 10° C.).

The systems described herein can optionally include a fluid conduitdisposed between the final chromatography column or chromatographicmembrane in the second MCCS and the outlet. The systems described hereincan further include one or more filters in fluid connection with thefluid conduit disposed between the final chromatography column orchromatographic membrane in the second MCCS and the outlet, such thatthe filter can remove, e.g., precipitated material, particulate matter,or bacteria from the fluid present in the fluid conduit disposed betweenthe final chromatography column or chromatographic membrane in thesecond MCCS and the outlet.

Some examples of the systems provided herein also include a bioreactorthat is in fluid connectivity with the inlet of the first MCCS. Any ofthe exemplary bioreactors described herein or known in the art can beused in the present systems.

Some examples of the systems provided herein also include a pump system.A pump system can include one or more the following: one or more (e.g.,two, three, four, five, six, seven, eight, nine, or ten) pumps (e.g.,any of the pumps described herein or known in the art), one or more(e.g., two, three, four, or five) filters (e.g., any of the filtersdescribed herein or known in the art), one or more (e.g., two, three,four, five, six, seven, eight, nine, or ten) UV detectors, and one ormore (e.g., two, three, four, or five) break tanks (e.g., any of thebreak tanks described herein). Some examples of the systems providedherein further include a fluid conduit disposed between the pump and theinlet of the first MCCS (e.g., any of the exemplary fluid conduitsdescribed herein or known in the art). In some examples, this particularfluid conduit can include one or more (e.g., two, three, or four) pumps(e.g., any of the pumps described herein or known in the art) and/or oneor more (e.g., two, three, or four) break tanks (e.g., any of theexemplary break tanks described herein), where these pump(s) and/orbreak tank(s) are in fluid connection with the fluid present in thefluid conduit.

Some examples of the systems described herein further include a furtherfluid conduit connected to the fluid conduit between the pump and theinlet, where one end of the further fluid conduit is fluidly connectedto a bioreactor and the other end is fluidly connected to the fluidconduit between the pump and the inlet. This further fluid conduit caninclude a filter that is capable of removing cells from the liquidculture medium removed from the bioreactor (e.g., ATF cell retentionsystem).

The systems provided herein allow for the continuous production of atherapeutic protein drug substance. For example, the systems providedherein allow for a percentage yield of recombinant therapeutic protein(from a starting material, e.g., a starting liquid culture medium) ofgreater than about 70%, greater than about 80%, greater than about 82%,greater than about 84%, greater than about 86%, greater than about 88%,greater than about 90%, greater than about 92%, greater than about 94%,greater than about 96%, or greater than about 98%. The systems describedherein can also result in a percentage yield of recombinant therapeuticprotein (from a starting material, e.g., a starting liquid culturemedium) of between about 80% to about 90%, between about 82% to about90%, between about 84% to about 90%, between about 84% to about 88%,between about 84% to about 94%, between about 82% to about 92%, orbetween about 85% to about 95%.

The systems described herein can also result in the production of atherapeutic protein drug substance that contains a concentration ofrecombinant therapeutic protein that is greater than about 1.0 mg/mL,greater than about 1.5 mg/mL, greater than about 2.0 mg/mL, greater thanabout 2.5 mg/mL, greater than about 3.0 mg/mL, greater than about 3.5mg/mL, greater than about 4.0 mg/mL, greater than about 4.5 mg/mL,greater than about 5.0 mg/mL, greater than about 5.5 mg/mL, greater thanabout 6.0 mg/mL, greater than about 6.5 mg/mL, greater than about 7.0mg/mL, greater than about 7.5 mg/mL, greater than about 8.0 mg/mL,greater than about 8.5 mg/mL, greater than about 9.0 mg/mL, greater thanabout 10.0 mg/mL, greater than about 12.5 mg/mL, or greater than about15.0 mg/mL. As is known in the art, the systems can provide for theperiodic elution of a therapeutic protein drug substance. Thetherapeutic protein drug substance can be eluted from any of the systemsdescribed herein for a duration of, e.g., between about 30 seconds andabout 5 hours (e.g., between about 1 minute and about 4 hours, betweenabout 1 minute and about 3 hours, between about 1 minute and about 2hours, between about 1 minute or about 1.5 hours, between about 1 minuteand about 1 hour, between about 1 minute and about 30 minutes) at afrequency of, e.g., between about 1 minute and about 6 hours (e.g.,between about 1 minute and about 5 hours, between about 1 minute andabout 4 hours, between about 1 minute and about 3 hours, between about 1minute and 2 hours, between about 1 minute and 1 hour, or between about1 minute and 30 minutes), depending on, e.g., the chromatographycolumn(s) and/or chromatographic membrane(s) used in the first andsecond MCCS.

The systems described herein can also result in a net yield ofrecombinant therapeutic protein in the therapeutic protein drugsubstance of at least about 5 g/day, at least about 6 g/day, at leastabout 7 g/day, at least about 8 g/day, at least about 9 g/day, at leastabout 10 g/day, at least about 11 g/day, at least about 12 g/day, atleast about 13 g/day, at least about 14 g/day, at least about 15 g/day,at least about 16 g/day, at least about 17 g/day, at least about 18g/day, at least about 19 g/day, at least about 20 g/day, at least about25 g/day, at least about 30 g/day, at least about 35 g/day, or at leastabout 40 g/day over a continuous period of at least about 5 days, atleast about 10 days, at least about 15 days, at least about 20 days, atleast about 25 days, at least about 30 days, at least about 35 days, atleast about 40 days, at least about 45 days, at least about 50 days, atleast about 55 days, at least about 60 days, at least about 65 days, atleast about 70 days, at least about 75 days, at least about 80 days, atleast about 85 days, at least about 90 days, at least about 95 days, atleast about 100 days, at least about 110 days, at least about 120 days,at least about 130 days, at least about 140 days, at least about 150days, at least about 160 days, at least about 170 days, at least about180 days, at least about 190 days, at least about 200 days, at leastabout 210 days, at least about 220 days, at least about 230 days, atleast about 240 days, at least about 250 days, at least about 260 days,at least about 270 days, at least about 280 days, at least about 290days, at least about 300 days, at least about 310 days, at least about320 days, at least about 330 days, at least about 340 days, at leastabout 350 days, or at least about 365 days.

The systems described herein can also continuously produce a recombinantprotein drug substance that contains recombinant therapeutic proteinhaving a significantly improved specific productivity rate (as comparedto other recombinant protein drug substances prepared by a differentprocess or system). For example, the present systems can achieve aspecific productivity rate (in the recombinant protein drug substance)that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold,80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold,150-fold, 160-fold, 170-fold, 180-fold, 190-fold, or 200-fold greaterthan the specific productivity rate in a recombinant protein drugsubstance produced using a different process or system (e.g., a batchpurification process or a process that is not integrated and/orcontinuous). The productivity in the recombinant protein drug substanceachieved by the present systems can be at least 10,000 units/L, at least15,000 units/L, at least about 20,000 units/L, at least about 25,000units/L, at least about 30,000 units/L, at least about 35,000 units/L,or at least about 40,000 units/L (in the first and/or second liquidculture medium). The productivity in the recombinant protein drugsubstance achieved by the present systems can be at least 1 g/L, atleast 1.5 g/L, at least 2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, atleast 4.0 g/L, at least 4.5 g/L, or at least 5.0 g/L.

Biological Manufacturing Systems with Two or More Subsystems

Also provided are biological manufacturing systems that include two ormore subsystems that each include: (i) a first multi-columnchromatography system (MCCS) containing an inlet (e.g., any of theexemplary first MCCSs described herein); and (ii) a second MCCScontaining an outlet (e.g., any of the exemplary second MCCSs describedherein), where the first and second MCCSs are in fluid communicationwith each other, and wherein the manufacturing system is configured suchthat fluid can be passed into the inlet, through the first and secondMCCS, and exit the manufacturing system through the outlet; where thetwo or more subsystems are configured such that they are each in fluidcommunication with a single reservoir containing a fluid (e.g., abioreactor), and the fluid from the single reservoir passes into theinlet of the two or more subsystems.

Each of the subsystems can provide for the continuous and time-efficientproduction of a therapeutic drug substance from a liquid culture medium.For example, the elapsed time for each subsystem, between feeding afluid (e.g., a liquid culture medium) containing a therapeutic proteininto the first MCCS (of the subsystem) and eluting a therapeutic proteindrug substance (containing the therapeutic protein) from the outlet ofthe second MCCS (of the subsystem) can be, e.g., between about 4 hoursand about 48 hours, inclusive, e.g., between about 4 hours and about 40hours, between about 4 hours and about 35 hours, between about 4 hoursand about 30 hours, between about 4 hours and about 28 hours, betweenabout 4 hours and about 26 hours, between about 4 hours and about 24hours, between about 4 hours and about 22 hours, between about 4 hoursand about 20 hours, between about 4 hours and about 18 hours, betweenabout 4 hours and about 16 hours, between about 4 hours and about 14hours, between about 4 hours and about 12 hours, between about 6 hoursand about 12 hours, between about 8 hours and about 12 hours, betweenabout 6 hours and about 20 hours, between about 6 hours and about 18hours, between about 6 hours and about 14 hours, between about 8 hoursand about 16 hours, between about 8 hours and about 14 hours, betweenabout 8 hours and about 12 hours, between about 10 hours and 20 hours,between about 10 hours and 18 hours, between about 10 hours and 16hours, between about 10 hours and 14 hours, between about 12 hours andabout 14 hours, between about 10 hours and about 40 hours, between about10 hours and about 35 hours, between about 10 hours and about 30 hours,between about 10 hours and about 25 hours, between about 15 hours andabout 40 hours, between about 15 hours and about 35 hours, between about15 hours and about 30 hours, between about 20 hours and about 40 hours,between about 20 hours and about 35 hours, or between about 20 hours andabout 30 hours, inclusive. In other examples, for each subsystem, theelapsed time between feeding the fluid (e.g., a liquid culture medium)containing a therapeutic protein into the first MCCS (of the subsystem)and eluting a therapeutic protein drug substance (containing thetherapeutic protein) from the outlet of the second MCCS (of thesubsystem) is, e.g., greater than about 4 hours and is less than about40 hours, inclusive, e.g., greater than about 4 hours and less thanabout 39 hours, about 38 hours, about 37 hours, about 36 hours, about 35hours, about 34 hours, about 33 hours, about 32 hours, about 31 hours,about 30 hours, about 29 hours, about 28 hours, about 27 hours, about 26hours, about 25 hours, about 24 hours, about 23 hours, about 22 hours,about 21 hours, about 20 hours, about 19 hours, about 18 hours, about 17hours, about 16 hours, about 15 hours, about 14 hours, about 13 hours,about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8hours, about 7 hours, about 6 hours, about 5 hours, or about 4.5 hours,inclusive.

Some exemplary systems subsystem(s) do not include a break tank. Othersinclude a maximum of 1, 2, 3, 4, or 5 break tanks in the entiresubsystem. Any of the subsystem(s) can include a maximum of 1, 2, 3, 4,or 5 break tank(s) in the entire subsystem, where each break tank onlyholds a therapeutic protein for a total time period of, e.g., betweenabout 5 minutes and less than about 6 hours, inclusive, e.g., betweenabout 5 minutes and about 5 hours, about 4 hours, about 3 hours, about 2hours, about 1 hour, or about 30 minutes, inclusive. Break tank(s)described herein can have a capacity that is, e.g., between 1 mL andabout 300 mL, inclusive, e.g., between 1 mL and about 280 mL, about 260mL, about 240 mL, about 220 mL, about 200 mL, about 180 mL, about 160mL, about 140 mL, about 120 mL, about 100 mL, about 80 mL, about 60 mL,about 40 mL, about 20 mL, or about 10 mL, inclusive. Any break tank(s)disposed in the subsystem such that fluid enters the break tank(s) priorto entering the MCCS1 (of the subsystem) can have a capacity that is,e.g., between 1 mL and about 100%, inclusive, e.g., between 1 mL andabout 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about 20%, about 10%, or about 5%, inclusive, of the loading volumeof the first column of the first MCCS (of the subsystem). Any breaktank(s) disposed in the subsystem such that fluid enters the breaktank(s) prior to entering the MCCS2 (in any of the subsystem(s)) (andafter the fluid exits the MCCS1 of the subsystem) can have a capacitythat is, e.g., between 1 mL and about 100%, inclusive, e.g., between 1mL and about 90%, about 80%, about 70%, about 60%, about 50%, about 40%,about 30%, about 20%, about 10%, or about 5%, inclusive, of the loadingvolume of the first column of the second MCCS (of the subsystem).

Any combination of the features (e.g., type and number of chromatographycolumns, type and number of chromatographic membranes, volume, size,resin, and unit operation(s), flow rates, pumps, in-line bufferadjustments, UV detectors, one or more filters, and column-switchingmechanisms) of the first MCCS and the second MCCS systems describedabove can be used in the first MCCS and second MCCS in the two or moresubsystems. For example, the first MCCS and second MCCS in the two ormore subsystems can be substantially the same. In other examples, thetwo or more subsystems are substantially the same as described in thesystems described the Examples.

Skids

Any of the biological manufacturing systems described herein can bedisposed on a skid. For example, a system including at least abioreactor, a first MCCS, and a second MCCS can be disposed on a singleskid. In other examples of any of the systems described herein, thefirst MCCS can be disposed on a first skid and the second MCCS can bedisposed on a second skid. Additional examples of any of the systemsdescribed herein include a first MCCS and a second MCCS disposed on asingle skid, or the entire system disposed on a single skid.

Some examples of the biological manufacturing systems described hereininclude two or more subsystems that each include: (i) a firstmulti-column chromatography system (MCCS) containing an inlet (e.g., anyof the exemplary first MCCSs described herein); and (ii) a second MCCScontaining an outlet (e.g., any of the exemplary second MCCSs describedherein), where the first and second MCCSs are in fluid communicationwith each other, and wherein the manufacturing system is configured suchthat fluid can be passed into the inlet, through the first and secondMCCS, and exit the manufacturing system through the outlet; where thetwo or more subsystems are configured such that they are each in fluidcommunication with a single reservoir containing a fluid (e.g., abioreactor), and the fluid from the single reservoir passes into theinlet of the two or more subsystems. In such systems, the entire systemcan be disposed on a skid; the reservoir and the one or more subsystemscan be on a single skid; each of the two or more subsystems is eachdisposed on its own skid; or the two or more subsystems can be disposedon a single skid. In any of the systems described herein, a reservoir(e.g., a plastic bag), a break tank (e.g., any of the break tanksdescribed herein), or a bioreactor containing a liquid culture mediumcontaining a recombinant therapeutic protein can be disposed on its ownskid.

Non-limiting examples of skids include two or more wheels, a roller, asled, or a ski that one or more structures that enable movement. As isappreciated by those skilled in the art, skids can be composed of anysolid material (e.g., wood, metal, or plastic). Suitable skids can beobtained from Wunderlich-Malec (Minnetonka, Minn.) and Renfrow Brothers(Spartenburg, S.C.). In systems that utilize more than one skid, theskids can be designed to fit together (e.g., fit together via a latch,turn-key, screw, or clamp device).

Integrated and Continuous Processes for Manufacturing a TherapeuticProtein Drug Substance

Provided herein are integrated and continuous processes formanufacturing a therapeutic protein drug substance. These processesinclude providing a liquid culture medium containing a recombinanttherapeutic protein that is substantially free of cells, where theliquid culture medium is fed into a first multi-column chromatographysystem (MCCS1); capturing the recombinant therapeutic protein in theliquid culture medium using the MCCS1, where the eluate of the MCCS1containing the recombinant therapeutic protein is continuously fed intoa second multi-column chromatography system (MCCS2); and purifying andpolishing the recombinant therapeutic protein using the MCCS2, where theeluate from the MCCS2 is a therapeutic protein drug substance, and theprocess is integrated and runs continuously from the liquid culturemedium to the eluate from the MCCS2 that is the therapeutic protein drugsubstance.

The processes described herein provide continuous and time-efficientproduction of a therapeutic drug substance from a liquid culture medium.For example, the elapsed time between feeding a fluid (e.g., a liquidculture medium) containing a therapeutic protein into the first MCCS andeluting a therapeutic protein drug substance (containing the therapeuticprotein) from the second MCCS can be, e.g., between about 4 hours andabout 48 hours, inclusive, e.g., between about 4 hours and about 40hours, between about 4 hours and about 35 hours, between about 4 hoursand about 30 hours, between about 4 hours and about 28 hours, betweenabout 4 hours and about 26 hours, between about 4 hours and about 24hours, between about 4 hours and about 22 hours, between about 4 hoursand about 20 hours, between about 4 hours and about 18 hours, betweenabout 4 hours and about 16 hours, between about 4 hours and about 14hours, between about 4 hours and about 12 hours, between about 6 hoursand about 12 hours, between about 8 hours and about 12 hours, betweenabout 6 hours and about 20 hours, between about 6 hours and about 18hours, between about 6 hours and about 14 hours, between about 8 hoursand about 16 hours, between about 8 hours and about 14 hours, betweenabout 8 hours and about 12 hours, between about 10 hours and 20 hours,between about 10 hours and 18 hours, between about 10 hours and 16hours, between about 10 hours and 14 hours, between about 12 hours andabout 14 hours, between about 10 hours and about 40 hours, between about10 hours and about 35 hours, between about 10 hours and about 30 hours,between about 10 hours and about 25 hours, between about 15 hours andabout 40 hours, between about 15 hours and about 35 hours, between about15 hours and about 30 hours, between about 20 hours and about 40 hours,between about 20 hours and about 35 hours, or between about 20 hours andabout 30 hours, inclusive. In other examples, the elapsed time betweenfeeding the fluid (e.g., a liquid culture medium) containing atherapeutic protein into the MCCS1 and eluting a therapeutic proteindrug substance (containing the therapeutic protein) from the MCCS2 is,e.g., greater than about 4 hours and less than about 40 hours,inclusive, e.g., greater than about 4 hours and less than about 39hours, about 38 hours, about 37 hours, about 36 hours, about 35 hours,about 34 hours, about 33 hours, about 32 hours, about 31 hours, about 30hours, about 29 hours, about 28 hours, about 27 hours, about 26 hours,about 25 hours, about 24 hours, about 23 hours, about 22 hours, about 21hours, about 20 hours, about 19 hours, about 18 hours, about 17 hours,about 16 hours, about 15 hours, about 14 hours, about 13 hours, about 12hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours,about 7 hours, about 6 hours, about 5 hours, or about 4.5 hours,inclusive.

Some exemplary processes do not utilize a holding step (e.g., do not usea reservoir (e.g., break tank) in the entire process). Others may use amaximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in theentire process. Any of the processes described herein can utilize amaximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) in theentire process, where each break tank only holds a therapeutic proteinfor a total time period of, e.g., between about 5 minutes and less thanabout 6 hours, inclusive, e.g., between about 5 minutes and about 5hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, orabout 30 minutes, inclusive. Any of the reservoir(s) (e.g., breaktank(s)) used in the processes described herein can have a capacity thatis, e.g., between 1 mL and about 300 mL, inclusive, e.g., between 1 mLand about 280 mL, about 260 mL, about 240 mL, about 220 mL, about 200mL, about 180 mL, about 160 mL, about 140 mL, about 120 mL, about 100mL, about 80 mL, about 60 mL, about 40 mL, about 20 mL, or about 10 mL(inclusive). Any reservoir(s) (e.g., break tank(s)) used (in any of theprocesses described herein) to hold fluid before it enters into thefirst MCCS can have a capacity that is, e.g., between 1 mL and about100%, inclusive, e.g., between 1 mL and about 90%, about 80%, about 70%,about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, orabout 5%, inclusive, of the loading volume of the first column of thefirst MCCS. Any of the reservoir(s) (e.g., break tanks(s)) used (in anyof the processes described herein) to hold fluid before it enters intothe second MCCS (and after the fluid leaves the first MCCS) can have acapacity that is, e.g., between 1 mL and about 100%, inclusive, e.g.,between 1 mL and about 90%, about 80%, about 70%, about 60%, about 50%,about 40%, about 30%, about 20%, about 10%, or about 5%, inclusive, ofthe loading volume of the first column of the second MCCS.

Various additional aspects of these processes are described in detailbelow and can be used in any combination in the processes providedherein without limitation. Exemplary aspects of the provided processesare described below; however, one skilled in the art will appreciatethat additional steps can be added to the processes described herein andother materials can be used to perform any of the steps of the processesdescribed herein.

Liquid Culture Medium

Liquid culture medium that contains a recombinant therapeutic proteinthat is substantially free of cells can be derived from any source. Forexample, the liquid culture medium can be obtained from a recombinantcell culture (e.g., a recombinant bacterial, yeast, or mammalian cellculture). The liquid culture medium can be obtained from a fed-batchcell (e.g., mammalian cell) culture (e.g., a fed-batch bioreactorcontaining a culture of mammalian cells that secrete the recombinanttherapeutic protein) or a perfusion cell (e.g., mammalian cell) culture(e.g., a perfusion bioreactor containing a culture of mammalian cellsthat secrete the recombinant therapeutic protein). The liquid culturemedium can also be a clarified liquid culture medium from a culture ofbacterial or yeast cells that secrete the recombinant therapeuticprotein.

Liquid culture medium obtained from a recombinant cell culture can befiltered or clarified to obtain a liquid culture medium that issubstantially free of cells and/or viruses. Methods for filtering orclarifying a liquid culture medium in order to remove cells are known inthe art (e.g., 0.2-μm filtration and filtration using an AlternatingTangential Flow (ATF™) system). Recombinant cells can also be removedfrom liquid culture medium using centrifugation and removing thesupernatant that is liquid culture medium that is substantially free ofcells, or by allowing the cells to settle to the gravitational bottom ofa container (e.g., bioreactor) containing the liquid culture medium, andremoving the liquid culture medium (the liquid culture medium that issubstantially free of cells) that is distant from the settledrecombinant cells.

The liquid culture medium can be obtained from a culture of recombinantcells (e.g., recombinant bacteria, yeast, or mammalian cells) producingany of the recombinant therapeutic proteins described herein. Someexamples of any of the processes described herein can further include astep of culturing recombinant cells (e.g., recombinant bacteria, yeast,or mammalian cells) that produce the recombinant therapeutic protein.

The liquid culture medium can be any of the types of liquid culturemedium described herein or known in the art. For example, the liquidculture medium can be selected from the group of: animal-derivedcomponent free liquid culture medium, serum-free liquid culture medium,serum-containing liquid culture medium, chemically-defined liquidculture medium, and protein-free liquid culture medium. In any of theprocesses described herein, a liquid culture medium obtained from aculture can be diluted by addition of a second fluid (e.g., a buffer)before it is fed into the first MCCS (e.g., first PCCS).

The liquid culture medium containing a recombinant therapeutic proteinthat is substantially free of cells can be stored (e.g., at atemperature below about 15° C. (e.g., below about 10° C., below about 4°C., below about 0° C., below about −20° C., below about −50° C., belowabout −70 C°, or below about −80° C.) for at least 1 day (e.g., at leastabout 2 days, at least about 5 days, at least about 10 days, at leastabout 15 days, at least about 20 days, or at least about 30 days) priorto feeding the liquid culture medium into the first MCCS (e.g., firstPCCS). Alternatively, in some examples the liquid culture medium is fedinto the first MCCS (e.g., first PCCS) directly from a bioreactor (e.g.,fed into the first MCCS (e.g., first PCCS) directly from the bioreactorafter a filtering or clarification step).

Recombinant Therapeutic Proteins

Non-limiting examples of recombinant therapeutic proteins that can beproduced by the methods provided herein include immunoglobulins(including light and heavy chain immunoglobulins, antibodies, orantibody fragments (e.g., any of the antibody fragments describedherein), enzymes (e.g., a galactosidase (e.g., an alpha-galactosidase),Myozyme, or Cerezyme), proteins (e.g., human erythropoietin, tumornecrosis factor (TNF), or an interferon alpha or beta), or immunogenicor antigenic proteins or protein fragments (e.g., proteins for use in avaccine). The recombinant therapeutic protein can be an engineeredantigen-binding polypeptide that contains at least one multifunctionalrecombinant protein scaffold (see, e.g., the recombinant antigen-bindingproteins described in Gebauer et al., Current Opin. Chem. Biol.13:245-255, 2009; and U.S. Patent Application Publication No.2012/0164066 (herein incorporated by reference in its entirety)).Non-limiting examples of recombinant therapeutic proteins that areantibodies include: panitumumab, omalizumab, abagovomab, abciximab,actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab,alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, amatuximab,anatumomab, anrukinzumab, apolizumab, arcitumomab, atinumab,tocilizumab, basilizimab, bectumomab, belimumab, bevacizumab,besilesomab, bezlotoxumab, biciromab, canakinumab, certolizumab,cetuximab, cixutumumab, daclizumab, denosumab, densumab, eculizumab,edrecolomab, efalizumab, efungumab, epratuzumab, ertumaxomab,etaracizumab, figitumumab, golimumab, ibritumomab tiuxetan, igovomab,imgatuzumab, infliximab, inolimomab, inotuzumab, labetuzumab,lebrikizumab, moxetumomab, natalizumab, obinutuzumab, oregovomab,palivizumab, panitumumab, pertuzumab, ranibizumab, rituximab,tocilizumab, tositumomab, tralokinumab, tucotuzumab, trastuzumab,veltuzumab, zalutumumab, and zatuximab. Additional examples ofrecombinant therapeutic antibodies that can be produced by the methodsdescribed herein are known in the art. Additional non-limiting examplesof recombinant therapeutic proteins that can be produced by the presentmethods include: alglucosidase alfa, laronidase, abatacept, galsulfase,lutropin alfa, antihemophilic factor, agalsidase beta, interferonbeta-1a, darbepoetin alfa, tenecteplase, etanercept, coagulation factorIX, follicle stimulating hormone, interferon beta-1a, imiglucerase,dornase alfa, epoetin alfa, insulin or insulin analogs, mecasermin,factor VIII, factor VIIa, anti-thrombin III, protein C, human albumin,erythropoietin, granulocute colony stimulating factor, granulocytemacrophage colony stimulating factor, interleukin-11, laronidase,idursuphase, galsulphase, α-1-proteinase inhibitor, lactase, adenosinedeaminase, tissue plasminogen activator, thyrotropin alpha (e.g.,Thyrogen®) and alteplase. Additional examples of recombinant proteinsthat can be produced by the present methods include acid α-glucosidase,alglucosidase alpha (e.g., Myozyme® and Lumizyme®), α-L-iduronidase(e.g., Aldurazyme®), iduronate sulfatase, heparan N-sulfatase,galactose-6-sulfatase, acid β-galactosidase, β-glucoronidase,N-acetylglucosamine-1-phosphotransferase, α-N-acetylgalactosaminidase,acid lipase, lysosomal acid ceramidase, acid sphingomyelinase,β-glucosidase (e.g., Cerezyme® and Ceredase®), galactosylceramidase,α-galactosidase-A (e.g., Fabrazyme®), acid β-galactosidase,β-galactosidase, neuraminidase, hexosaminidase A, and hexosaminidase B.

A secreted, soluble recombinant therapeutic protein can be recoveredfrom the liquid culture medium (e.g., a first and/or second liquidculture medium) by removing or otherwise physically separating theliquid culture medium from the cells (e.g., mammalian cells). A varietyof different methods for removing liquid culture medium from cells(e.g., mammalian cells) are known in the art, including, for example,centrifugation, filtration, pipetting, and/or aspiration. The secretedrecombinant therapeutic protein can then be recovered and furtherpurified from the liquid culture medium using a variety of biochemicaltechniques including various types of chromatography (e.g., affinitychromatography, molecular sieve chromatography, cation exchangechromatography, or anion exchange chromatography) and/or filtration(e.g., molecular weight cut-off filtration).

Multi-Column Chromatography Systems

The processes described herein include the use of two or more (e.g.,two, three, four, five, or six) multi-column chromatography systems(MCCSs). A MCCS can include two or more chromatography columns, two ormore chromatographic membranes, or a combination of at least onechromatography column and at least one chromatographic membrane. Innon-limiting examples, a MCCS (e.g., the first and/or second MCCSdescribed in any of the processes herein) can include fourchromatographic columns, three chromatographic columns and achromatographic membrane, three chromatographic columns, twochromatographic columns, two chromatographic membranes, and twochromatographic columns and one chromatographic membrane. Additionalexamples of combinations of chromatography columns and/orchromatographic membranes can be envisioned for use in an MCCS (e.g., afirst and/or second MCCS) by one skilled in the art without limitation.The individual chromatography columns and/or chromatographic membranespresent in a MCCS can be identical (e.g., have the same shape, volume,resin, capture mechanism, and unit operation), or can be different(e.g., have one or more of a different shape, volume, resin, capturemechanism, and unit operation). The individual chromatography column(s)and/or chromatographic membrane(s) present in a MCCS (e.g., the firstand/or second MCCS) can perform the same unit operation (e.g., the unitoperation of capturing, purifying, or polishing) or different unitoperations (e.g., different unit operations selected from, e.g., thegroup of capturing, purifying, polishing, inactivating viruses,adjusting the ionic concentration and/or pH of a fluid containing therecombinant therapeutic protein, and filtering).

The one or more chromatography column(s) that can be present in an MCCS(e.g., present in the first and/or second MCCS) can have a resin volumeof, e.g., at least about 2 mL, at least about 5 mL, at least about 10mL, at least about 15 mL, at least about 20 mL, at least about 25 mL, atleast about 30 mL, at least about 35 mL, at least about 40 mL, at leastabout 45 mL, at least about 50 mL, at least about 55 mL, at least about60 mL, at least about 65 mL, at least about 70 mL, at least about 75 mL,at least about 80 mL, at least about 85 mL, at least about 90 mL, atleast about 95 mL, or at least about 100 mL. The one or morechromatography column(s) that can be present in an MCCS (e.g., presentin the first and/or second MCCS) can have a resin volume of betweenabout 2 mL to about 100 mL, between about 2 mL and about 90 mL, betweenabout 2 mL and about 80 mL, between about 2 mL and about 70 mL, betweenabout 2 mL and about 60 mL, between about 2 mL and about 50 mL, betweenabout 5 mL and about 50 mL, between about 2 mL and about 45 mL, betweenabout 5 mL and about 45 mL, between about 2 mL and about 40 mL, betweenabout 5 mL and about 40 mL, between about 2 mL and about 35 mL, betweenabout 5 mL and about 35 mL, between about 2 mL and about 30 mL, betweenabout 5 mL and about 30 mL, between about 2 mL and about 25 mL, betweenabout 5 mL and about 25 mL, between about 15 mL and about 60 mL, betweenabout 10 mL and about 60 mL, between about 10 mL and about 50 mL, andbetween about 15 mL and about 50 mL. The one or more chromatographycolumn(s) in an MCCS (e.g., the first and/or second MCCS) used in any ofthe processes described herein can have the substantially the same resinvolume or can have different resin volumes. The flow rate used for theone or more chromatography column(s) in an MCCS (e.g., the first and/orsecond MCCS) can be, e.g., between about 0.2 mL/minute to about 25mL/minute (e.g., between about 0.2 mL/minute to about 20 mL/minute,between about 0.5 mL/minute to about 20 mL/minute, between about 0.2mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 10 mL/minute, betweenabout 0.5 mL minute and about 14 mL/minute, between about 1.0 mL/minuteand about 25.0 mL/minute, between about 1.0 mL/minute and about 15.0mL/minute).

The one or more chromatography column (s) in an MCCS (e.g., the firstand/or second MCCS) can have substantially the same shape or can havesubstantially different shapes. For example, the one or morechromatography column(s) in an MCCS (e.g., in the first and/or secondMCCS) can have substantially the shape of a circular cylinder or canhave substantially the same shape of an oval cylinder.

The one or more chromatographic membrane(s) that can be present in anMCCS (e.g., present in the first and/or second MCCS) can have a bedvolume of, e.g., between about 1 mL to about 500 mL (e.g., between about1 mL to about 475 mL, between about 1 mL to about 450 mL, between about1 mL to about 425 mL, between about 1 mL to about 400 mL, between about1 mL to about 375 mL, between about 1 mL to about 350 mL, between about1 mL to about 325 mL, between about 1 mL to about 300 mL, between about1 mL to about 275 mL, between about 1 mL to about 250 mL, between about1 mL to about 225 mL, between about 1 mL to about 200 mL, between about1 mL to about 175 mL, between about 1 mL to about 150 mL, between about1 mL to about 125 mL, between about 1 mL to about 100 mL, between about2 mL to about 100 mL, between about 5 mL to about 100 mL, between about1 mL to about 80 mL, between about 2 mL to about 80 mL, between about 5mL to about 80 mL, between about 1 mL to about 60 mL, between about 2 mLto about 60 mL, between about 5 mL to about 60 mL, between about 1 mL toabout 40 mL, between about 2 mL to about 40 mL, between about 5 mL toabout 40 mL, between about 1 mL to about 30 mL, between about 2 mL toabout 30 mL, between about 5 mL to about 30 mL, between about 1 mL andabout 25 mL, between about 2 mL and about 25 mL, between about 1 mL andabout 20 mL, between about 2 mL and about 20 mL, between about 1 mL andabout 15 mL, between about 2 mL and about 15 mL, between about 1 mL andabout 10 mL, or between about 2 mL and about 10 mL.

One or more (e.g., three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, ortwenty-four) different types of buffer can be employed during the use ofthe two or more MCCSs in any of the processes described herein. As isknown in the art, the one or more types of buffer used in the two ormore MCCSs used in the processes described herein will depend on theresin present in the chromatography column(s) and/or the chromatographicmembrane(s) of the two or more MCCSs (e.g., the first and second MCCSs),the recombinant therapeutic protein, and unit operation (e.g., any ofthe exemplary unit operations described herein) performed by thespecific chromatography column(s) and/or chromatography membranes of thetwo or more MCCSs. The volume and type of buffer employed during the useof the two or more MCCSs in any of the processes described herein canalso be determined by one skilled in the art (e.g., discussed in moredetail below). For example, the volume and type(s) of buffer employedduring the use of the two or more MCCSs in any of the processesdescribed herein can be chosen in order to optimize one or more of thefollowing in the recombinant protein drug product: the overall yield ofrecombinant therapeutic protein, the activity of the recombinanttherapeutic protein, the level of purity of the recombinant therapeuticprotein, and the removal of biological contaminants from a fluidcontaining the recombinant therapeutic protein (e.g., absence of activeviruses, mycobacteria, yeast, bacteria, or mammalian cells).

The first and/or second MCCS can be a periodic counter currentchromatography system (PCCS). A PCCS can, e.g., include two or morechromatography columns (e.g., three columns or four columns) that areswitched in order to allow for the continuous elution of recombinanttherapeutic protein from the two or more chromatography columns. A PCCScan include two or more chromatography columns, two or morechromatographic membranes, or at least one chromatographic column and atleast one chromatographic membrane. A column operation generallyconsists of the load, wash, eluate, and regeneration steps. In PCCSs,multiple columns are used to run the same steps discretely andcontinuously in a cyclic fashion. Since the columns are operated inseries, the flow through and wash from one column is captured by anothercolumn. This unique feature of PCCSs allows for loading of the resinclose to its static binding capacity instead of to the dynamic bindingcapacity, as is typical during batch mode chromatography. An example ofthe three column-switching technique used in a PCCS containing threecolumns is shown in FIG. 3. A cycle is defined as three complete columnoperations resulting in an elution pool from each of the three columnsused in the column-switching technique. Once all the steps in the cycleare completed, the cycle is re-started. As a result of the continuouscycling and elutation, fluid entering a PCCS is processed continuously,and the eluate containing recombinant therapeutic protein iscontinuously produced.

To advance from one step to another in a PCCS cycle, such as theexemplary cycle shown in FIG. 3, a column-switching strategy isemployed. The column switching method employs two automated switchingoperations per column in the three-columns in the exemplary PCCS systemshown in FIG. 3, the first of which is related to the initial productbreakthrough, while the second coincides with column saturation. Thedetermination of when the column switching operations should take placeis determined by monitoring the recombinant therapeutic proteinconcentration (e.g., monitoring performed by UV monitoring) in theeluate from each chromatography column present in a PCCS. For example,column switching can be determined by any PAT tool capable of in-linemeasurement of product concentration with feedback control. The PAT toolis capable of real-time in-line measurement of product concentrationwith feedback control.

FIG. 4 depicts an example of column switching in an exemplary PCCS basedon the UV absorbance difference (ΔUV) between the feed inlet and columnoutlet. For example, during column loading (Step 1; FIG. 3), the PCCcontrol system determines the impurity baseline level when theabsorbance stabilizes. As the product breaks through (Step 2; FIG. 3),there is an increase in the outlet UV signal above the impuritybaseline. At the point when ΔUV has reached a pre-determined threshold(e.g., 3% breakthrough of the product), the flow-through from column 1is directed onto column 2 instead of to the waste (t1; FIG. 4). Whencolumn 1 is nearly saturated with product and the ΔUV has reached apre-determined value (t2; FIG. 4), the feed is switched to column 2. Thecolumn-switching strategy used in PCCSs allows for the uniform loadingof the columns irrespective of the feed product concentration and thecapacity. Similar switches of the columns based on the level ofrecombinant protein detected in the eluate from each column can bedesigned. As in known in the art, column switches can also be designedbased on time or the amount of fluid (e.g., buffer) passed through theone or more chromatography column(s) and/or chromatographic membranes inthe first or second MCCS.

In PCCSs, the residence time (RT) of the recombinant therapeutic proteinon the each chromatography column and/or chromatographic membranepresent in the PCCS can be decreased without increasing thecolumn/membrane size because the breakthrough from the firstcolumn/membrane can be captured on another column/membrane in the PCCS.A continuous process system can be designed to process liquid culturemedium at any perfusion rate (D) by varying the column/membrane volume(V) and RT using the equation of: V=D*RT.

The one or more unit operations that can be performed by the at leasttwo MCCSs (e.g., the first and/or second MCCSs) used in the presentlydescribed processes include, for example, capturing the recombinanttherapeutic protein, inactivating viruses present in a fluid containingthe recombinant therapeutic protein, purifying the recombinanttherapeutic protein, polishing the recombinant therapeutic protein,holding a fluid containing the recombinant therapeutic protein (e.g.,using any of the exemplary break tank(s) described herein), filtering orremoving particulate material and/or cells from a fluid containing therecombinant therapeutic protein, and adjusting the ionic concentrationand/or pH of a fluid containing the recombinant therapeutic protein.

The unit operation of capturing can be performed using one or more MCCSs(e.g., a first and/or second MCCS) that includes at least onechromatography column and/or chromatography resin, e.g., that utilizes acapture mechanism. Non-limiting examples of capturing mechanisms includea protein A-binding capture mechanism, an antibody- or antibodyfragment-binding capture mechanism, a substrate-binding capturemechanism, an aptamer-binding capture mechanism, a tag-binding capturemechanism (e.g., poly-His tag-based capture mechanism), and acofactor-binding capture mechanism. Capturing can also be performedusing a resin that can be used to perform cation exchange or anionexchange chromatography, or molecular sieve chromatography. Non-limitingresins that can be used to capture a recombinant therapeutic protein aredescribed herein. Additional examples of resins that can be used tocapture a recombinant therapeutic protein are known in the art.

The unit operation of inactivating viruses present in a fluid containingthe recombinant therapeutic protein can be performed using one or moreMCCSs (e.g., a first and/or second MCCS) that include(s), e.g., achromatography column, a chromatography membrane, or a holding tank thatis capable of incubating a fluid containing the recombinant therapeuticprotein at a pH of between about 3.0 to 5.0 (e.g., between about 3.5 toabout 4.5, between about 3.5 to about 4.25, between about 3.5 to about4.0, between about 3.5 to about 3.8, or about 3.75) for a period of atleast 30 minutes (e.g., a period of between about 30 minutes to 1.5hours, a period of between about 30 minutes to 1.25 hours, a period ofbetween about 0.75 hours to 1.25 hours, or a period of about 1 hour).

The unit operation of purifying a recombinant protein can be performedusing one or more MCCSs (e.g., a first and/or second MCCS) thatinclude(s), e.g., a chromatography column or chromatographic membranethat contains a resin, e.g., that utilizes a capture system.Non-limiting examples of capturing mechanisms include a proteinA-binding capture mechanism, an antibody- or antibody fragment-bindingcapture mechanism, a substrate-binding capture mechanism, anaptamer-binding capture mechanism, a tag-binding capture mechanism(e.g., poly-His tag-based capture mechanism), and a cofactor-bindingcapture mechanism. Purifying can also be performed using a resin thatcan be used to perform cation exchange or anion exchange chromatography,or molecular sieve chromatography. Non-limiting resins that can be usedto purify a recombinant therapeutic protein are described herein.Additional examples of resins that can be used to purify a recombinanttherapeutic protein are known in the art.

The unit operation of polishing a recombinant protein can be performedusing one or more MCCSs (e.g., a first and/or second MCCS) thatinclude(s), e.g., a chromatography column or chromatographic membranethat contains a resin, e.g., that can be used to perform cationexchange, anion exchange, or molecular sieve chromatography.Non-limiting resins that can be used to polish a recombinant therapeuticprotein are described herein. Additional examples of resins that can beused to polish a recombinant therapeutic protein are known in the art.

The unit operation of holding a fluid containing the recombinanttherapeutic protein can be performed using an MCCS (e.g., a first and/orsecond MCCS) that includes at least one reservoir (e.g., a break tank)or a maximum of 1, 2, 3, 4, or 5 reservoir(s) (e.g., break tank(s)) inthe first and second MCCS combined. For example, the reservoir(s) (e.g.,break tank(s)) that can be used to achieve this unit operation can eachhave a volume of between about 1 mL to about 1 L (e.g., between about 1mL to about 800 mL, between about 1 mL to about 600 mL, between about 1mL to about 500 mL, between about 1 mL to about 400 mL, between about 1mL to about 350 mL, between about 1 mL to about 300 mL, between about 10mL and about 250 mL, between about 10 mL and about 200 mL, between about10 mL and about 150 mL, and between about 10 mL to about 100 mL). Thereservoir(s) (e.g., break tank(s)) used in the processes describedherein can have a capacity that is, e.g., between 1 mL and about 300 mL,inclusive, e.g., between 1 mL and about 280 mL, about 260 mL, about 240mL, about 220 mL, about 200 mL, about 180 mL, about 160 mL, about 140mL, about 120 mL, about 100 mL, about 80 mL, about 60 mL, about 40 mL,about 20 mL, or about 10 mL, inclusive. Any of the reservoir(s) (e.g.,break tank(s)) used (in any of the processes described herein) to holdfluid before it enters into the first MCCS can have a capacity that is,e.g., between 1 mL and about 100%, inclusive, between about 1 mL andabout 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about30%, about 20%, about 10%, or about 5%, inclusive, of the loading volumeof the first column of the first MCCS. Any of the reservoir(s) (e.g.,break tanks(s)) used to hold a fluid before it enters the second MCCS(and after the fluid leaves the first MCCS) can have a capacity that is,e.g., between 1 mL and about 100%, inclusive, e.g., between about 1 mLand about 90%, about 80%, about 70%, about 60%, about 50%, about 40%,about 30%, about 20%, about 10%, or about 5%, inclusive, of the loadingvolume of the first column of the second MCCS.

The reservoir(s) (e.g., break tank(s)) can each hold the fluidcontaining the recombinant therapeutic protein for at least 10 minutes(e.g., at least 20 minutes, at least 30 minutes, at least 1 hour, atleast 2 hours, at least 4 hours, or at least 6 hours). In otherexamples, the reservoir(s) (e.g., break tank(s)) only holds atherapeutic protein for a total time period of, e.g., between about 5minutes and less than about 6 hours, inclusive, e.g., between about 5minutes and about 5 hours, about 4 hours, about 3 hours, about 2 hours,about 1 hour, or about 30 minutes, inclusive. The reservoir(s) (e.g.,break tank(s)) can be used to both hold and refrigerate (e.g., at atemperature of less than 25° C., less than 15° C., or less than 10° C.)the fluid containing the recombinant therapeutic protein. The reservoircan have any shape, including a circular cylinder, an oval cylinder, oran approximately rectangular sealed and nonpermeable bag.

The unit operations of filtering a fluid containing the recombinanttherapeutic protein can be performed using an MCCS (e.g., the firstand/or second MCCS) that includes, e.g., a filter, or a chromatographycolumn or chromatographic membrane that contains a molecule sieve resin.As is known in the art, a wide variety of submicron filters (e.g., afilter with a pore size of less than 1 μm, less than 0.5 μm, less than0.3 μm, about 0.2 μm, less than 0.2 μm, less than 100 nm, less than 80nm, less than 60 nm, less than 40 nm, less than 20 nm, or less than 10nm) are available in the art that are capable of removing anyprecipitated material and/or cells (e.g., precipitated, unfoldedprotein; precipitated, unwanted host cell proteins; precipitated lipids;bacteria; yeast cells; fungal cells; mycobacteria; and/or mammaliancells). Filters having a pore size of about 0.2 μm or less than 0.2 μmare known to effectively remove bacteria from the fluid containing therecombinant therapeutic protein. As is known in the art, achromatography column or a chromatographic membrane containing amolecular sieve resin can also be used in an MCCS (e.g., the firstand/or second MCCS) to perform the unit operation of filtering a fluidcontaining a recombinant therapeutic protein.

The unit operations of adjusting the ionic concentration and/or pH of afluid containing the recombinant therapeutic protein can be performedusing a MCCS (e.g., the first and/or second MCCS) that includes andutilizes a buffer adjustment reservoir (e.g., an in-line bufferadjustment reservoir) that adds a new buffer solution into a fluid thatcontains the recombinant therapeutic protein (e.g., between columnswithin a single MCCS, or after the last column in a penultimate MCCS(e.g., the first MCCS) and before the fluid containing the recombinanttherapeutic protein is fed into the first column of the next MCCS (e.g.,the second MCCS). As can be appreciated in the art, the in-line bufferadjustment reservoir can be any size (e.g., greater than 100 mL) and cancontain any buffered solution (e.g., a buffered solution that has one ormore of: an increased or decreased pH as compared to the fluidcontaining the recombinant therapeutic protein, a an increased ordecreased ionic (e.g., salt) concentration compared to the fluidcontaining the recombinant therapeutic protein, and/or an increased ordecreased concentration of an agent that competes with the recombinanttherapeutic protein for binding to resin present in at least onechromatographic column or at least one chromatographic membrane in anMCCS (e.g., the first or the second MCCS)).

The first and/or second MCCS can perform two or more unit operations.For example, the first and/or second MCCS can each perform at least thefollowing unit operations: capturing the recombinant therapeutic proteinand inactivating viruses present in the fluid containing the recombinanttherapeutic protein; capturing the recombinant therapeutic protein,inactivating viruses present in the fluid containing the recombinanttherapeutic protein, and adjusting the ionic concentration and/or pH ofa liquid containing the recombinant therapeutic protein; purifying therecombinant therapeutic protein and polishing the recombinanttherapeutic protein; purifying the recombinant therapeutic protein,polishing the recombinant therapeutic protein, and filtering a fluidcontaining the recombinant therapeutic protein or removing precipitatesand/or particular matter from a fluid containing the recombinanttherapeutic protein; and purifying the recombinant therapeutic protein,polishing the recombinant therapeutic protein, filtering a fluidcontaining the recombinant therapeutic protein or removing precipitatesand/or particular matter from a fluid containing the recombinanttherapeutic protein, and adjusting the ionic concentration and/or pH ofa liquid containing the recombinant therapeutic protein.

Capturing the Recombinant Therapeutic Protein

The present processes include a step of capturing the recombinanttherapeutic protein using a first MCCS. As can be appreciated in theart, the liquid culture medium containing the recombinant therapeuticprotein can be continuously fed onto the first MCCS using a variety ofdifferent means. For example, the liquid culture medium can be activelypumped into the first MCCS or the liquid culture medium can be fed intothe first MCCS using gravitational force. The liquid culture medium canbe stored in a reservoir (e.g., a holding tank) before it is fed intothe first MCCS or the liquid culture medium can be actively pumped froma bioreactor containing a culture of cells (e.g., mammalian cells thatsecrete the recombinant therapeutic protein into the culture medium)into the first MCCS.

The liquid culture medium can be fed (loaded) into the first MCCS at aflow rate of between about 0.2 mL/minute to about 25 mL/minute (e.g.,between about 0.2 mL/minute to about 20 mL/minute, between about 0.5mL/minute to about 20 mL/minute, between about 0.2 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 15 mL/minute, betweenabout 0.5 mL/minute to about 10 mL/minute, between about 0.5 mL minuteand about 14 mL/minute, between about 1.0 mL/minute and about 25.0mL/minute, between about 1.0 mL/minute and about 15.0 mL/minute). Theliquid culture medium containing the recombinant therapeutic protein canbe derived from any of the exemplary sources described herein or knownin the art.

Some examples further include the optional step of filtering the liquidculture medium before it is fed onto the first MCCS. Any of theexemplary means of filtering a liquid culture medium or a fluidcontaining the recombinant therapeutic protein described herein, or anyfiltration means known in the art, can be used to filter the liquidculture medium containing the recombinant therapeutic protein before itis fed into the first MCCS.

In the methods described herein, the capturing of the recombinanttherapeutic protein from the liquid culture medium is performed using afirst MCCS. As can be appreciated in the art, in order to achieve thecapture of the recombinant therapeutic protein, at least onechromatographic column or at least one chromatographic membrane in thefirst MCCS must contain a resin that utilizes a capturing mechanism(e.g., any of the exemplary capturing mechanisms described herein), orcontains a resin capable of performing cation exchange, anion exchange,or molecule sieve chromatography. For example, if the recombinanttherapeutic protein is an antibody or an antibody fragment, thecapturing system can be a protein A-binding capturing mechanism or anantigen-binding capturing mechanism (where the capturing antigen isspecifically recognized by the recombinant therapeutic antibody orantibody fragment). If the recombinant therapeutic protein is an enzyme,the capturing mechanism can use an antibody or antibody fragment thatspecifically binds to the enzyme to capture the recombinant therapeuticenzyme, a substrate of the enzyme to capture the recombinant therapeuticenzyme, a cofactor of the enzyme to capture the recombinant therapeuticenzyme, or, if the recombinant therapeutic enzyme contains a tag, aprotein, metal chelate, or antibody (or antibody fragment) thatspecifically binds to the tag present in the recombinant therapeuticenzyme. Non-limiting resins that can be used to capture a recombinanttherapeutic protein are described herein and additional resins that canbe used to capture a recombinant therapeutic protein are known in theart. One non-limiting example of resin that utilizes a protein A-bindingcapture mechanism is MabSelect SuRe resin (GE Healthcare, Piscataway,N.J.).

Exemplary non-limiting sizes and shapes of the chromatography column(s)or chromatographic membrane(s) present in the first MCCS that can beused to capture the recombinant therapeutic protein are describedherein. The liquid culture medium fed (loaded) into the first MCCS cancontain, e.g., between about 0.05 mg/mL to about 100 mg/mL recombinanttherapeutic protein (e.g., between about 0.1 mg/mL to about 90 mg/mL,between about 0.1 mg/mL to about 80 mg/mL, between about 0.1 mg/mL toabout 70 mg/mL, between about 0.1 mg/mL to about 60 mg/mL, between about0.1 mg/mL to about 50 mg/mL, between about 0.1 mg/mL to about 40 mg/mL,between about 0.1 mg/mL to about 30 mg/mL, between about 0.1 mg/mL toabout 20 mg/mL, between 0.5 mg/mL to about 20 mg/mL, between about 0.1mg/mL to about 15 mg/mL, between about 0.5 mg/mL to about 15 mg/mL,between about 0.1 mg/mL to about 10 mg/mL, or between about 0.5 mg/mL toabout 10 mg/mL recombinant therapeutic protein). The mean time requiredfor the recombinant therapeutic protein to bind to the resin used toperform the unit operation of capturing can be, e.g., between about 5seconds to about 10 minutes (e.g., between about 10 seconds to about 8minutes, between about 10 seconds to about 7 minutes, between about 10seconds to about 6 minutes, between about 10 seconds to about 5 minutes,between about 30 seconds to about 5 minutes, between about 1 minute toabout 5 minutes, between about 10 seconds to about 4 minutes, betweenabout 30 seconds to about 4 minutes, or between about 1 minute to about4 minutes).

As can be appreciated in the art, in order to capture the recombinanttherapeutic protein using the chromatography column(s) orchromatographic membrane(s) present in the first MCCS, one must performthe sequential chromatographic steps of loading, washing, eluting, andregenerating the chromatography column(s) or chromatography membrane(s)present in the first MCCS. Any of the exemplary flow rates, buffervolumes, and/or lengths of time allotted for each sequentialchromatographic step described herein can be used in the one or more ofthese different sequential chromatographic steps (e.g., one or more ofthe sequential chromatographic steps of loading, washing, eluting, andregenerating the chromatography column(s) or chromatography membrane(s)present in the first MCCS that are used for capturing the recombinanttherapeutic protein). Non-limiting flow rates, buffer volumes, and/orlengths of time allotted for each sequential chromatographic step thatcan be used for capturing chromatographic column(s) and/orchromatographic membrane(s) in the first MCCS (e.g., a first PCC system)are provided below. In addition, exemplary buffers elution buffers thatcan be used in the first MCCS are described below.

The first MCCS containing at least one chromatographic column and/orchromatographic membrane containing a resin that can perform the unitoperation of capturing (e.g., any of exemplary resins that can be usedfor capturing described herein) can be loaded with the liquid culturemedium containing a recombinant therapeutic protein using any of loadingflow rates (fed rates) described above. In some examples, a singlechromatographic column or single chromatographic membrane containing aresin that is capable of performing the unit operation of capturing isloaded in, e.g., between about 10 minutes to about 90 minutes (e.g.,between about 15 minutes and about 90 minutes, between about 20 minutesand 80 minutes, between about 30 minutes and 80 minutes, between about40 minutes and about 80 minutes, between about 50 minutes and about 80minutes, and between about 60 minutes and 80 minutes). In some examples,wherein the first MCCS includes at least two chromatographic columnsthat contain a resin that is capable of performing the unit operation ofcapturing in series, the time required to load two of thechromatographic columns in series is, e.g., between about 50 minutes toabout 180 minutes (e.g., between about 60 minutes and about 180 minutes,between about 70 minutes and about 180 minutes, between about 80 minutesand about 180 minutes, between about 90 minutes and about 180 minutes,between about 100 minutes and about 180 minutes, between about 110minutes and 150 minutes, and between about 125 minutes and about 145minutes).

Following the loading of the recombinant therapeutic protein onto the atleast one chromatographic column or chromatographic membrane in thefirst MCCS that contains a resin that is capable of performing the unitoperation of capturing, the at least one chromatographic column orchromatographic membrane is washed with at least one washing buffer. Ascan be appreciated in the art, the at least one (e.g., two, three, orfour) washing buffer is meant to elute all proteins that are not therecombinant therapeutic protein from the at least one chromatographycolumn or chromatographic membrane, while not disturbing the interactionof the recombinant therapeutic protein with the resin.

The wash buffer can be passed through the at least one chromatographycolumn or chromatographic membrane at a flow rate of between about 0.2mL/minute to about 25 mL/minute (e.g., between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 1.0mL/minute and about 15.0 mL/minute). The volume of wash buffer used(e.g., combined total volume of wash buffer used when more than one washbuffer is used) can be, e.g., between about 1× column volume (CV) toabout 15×CV (e.g., between about 1×CV to about 14×CV, about 1×CV toabout 13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about2×CV to about 11×CV, about 3×CV to about 11×CV, about 4×CV to about11×CV, about 5×CV to about 11×CV, or about 5×CV to about 10×CV). Thetotal time of the washing can be, e.g., between about 2 minutes to about3 hours (e.g., between about 2 minutes to about 2.5 hours, between about2 minutes to about 2.0 hours, between about 5 minutes to about 1.5hours, between about 10 minutes to about 1.5 hours, between about 10minutes to about 1.25 hours, between about 20 minutes to about 1.25hours, or between about 30 minutes to about 1 hour).

Following the washing of the at least one chromatographic column orchromatographic membrane in the first MCCS that contains a resin that iscapable of performing the unit operation of capturing, the recombinanttherapeutic protein is eluted from the at least one chromatographiccolumn or chromatographic membrane by passing an elution buffer throughthe at least one chromatographic column or chromatographic membrane inthe first MCCS that contains a resin that is capable of performing theunit operation of capturing. The elution buffer can be passed throughthe at least one chromatography column or chromatographic membrane thatcontains a resin that is capable of performing the unit operation ofcapturing at a flow rate of between about 0.2 mL/minute to about 25mL/minute (e.g., between about 0.2 mL/minute to about 20 mL/minute,between about 0.5 mL/minute to about 20 mL/minute, between about 0.2mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 10 mL/minute, betweenabout 0.5 mL/minute and about 6.0 mL/minute, between about 1.0 mL/minuteand about 5.0 mg/minute, between about 0.5 mL minute and about 14mL/minute, between about 1.0 mL/minute and about 25.0 mL/minute, betweenabout 1.0 mL/minute and about 15.0 mL/minute). The volume of elutionbuffer used to elute the recombinant therapeutic protein from each ofthe at least one chromatographic column or chromatographic membranecontaining a resin that is capable of performing the unit operation ofpurifying can be, e.g., between about 1× column volume (CV) to about15×CV (e.g., between about 1×CV to about 14×CV, about 1×CV to about13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about 2×CVto about 11×CV, about 3×CV to about 11×CV, about 4×CV to about 11×CV,about 5×CV to about 11×CV, or about 5×CV to about 10×CV). The total timeof the eluting can be, e.g., between about 2 minutes to about 3 hours(e.g., between about 2 minutes to about 2.5 hours, between about 2minutes to about 2.0 hours, between about 2 minutes to about 1.5 hours,between about 2 minutes to about 1.5 hours, between about 2 minutes toabout 1.25 hours, between about 2 minutes to about 1.25 hours, betweenabout 2 minutes to about 1 hour, between about 2 minutes and about 40minutes, between about 10 minutes and about 40 minutes, between about 20minutes and about 40 minutes). Non-limiting examples of elution buffersthat can be used in these methods will depend on the capture mechanismand/or the therapeutic protein. For example, an elution buffer cancontain a different concentration of salt (e.g., increased saltconcentration), a different pH (e.g., an increased or decreased saltconcentration), or a molecule that will compete with the recombinanttherapeutic protein for binding to the resin that is capable ofperforming the unit operation of capturing. Examples of such elutionbuffers for each exemplary capture mechanism described herein are wellknown in the art.

Following the elution of the recombinant therapeutic protein from the atleast one chromatographic column or chromatographic membrane in thefirst MCCS that contains a resin that is capable of performing the unitoperation of capturing, and before the next volume of liquid culturemedium can be loaded onto the at least one chromatographic column orchromatographic membrane, the at least one chromatography column orchromatographic membrane must be equilibrated using an regenerationbuffer. The regeneration buffer can be passed through the at least onechromatography column or chromatographic membrane that contains a resinthat is capable of performing the unit operation of capturing at a flowrate of, e.g., between about 0.2 mL/minute to about 25 mL/minute (e.g.,between about 0.2 mL/minute to about 20 mL/minute, between about 0.5mL/minute to about 20 mL/minute, between about 0.2 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 15 mL/minute, betweenabout 0.5 mL/minute to about 10 mL/minute, between about 0.5 mL/minuteand about 6.0 mL/minute, between about 1.0 mL/minute and about 5.0mg/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 5.0mL/minute to about 15.0 mL/minute, or between about 1.0 mL/minute andabout 15.0 mL/minute). The volume of regeneration buffer used toequilibrate the at least one chromatography column or chromatographicmembrane that contains a resin that is capable of performing the unitoperation of capturing can be, e.g., between about 1× column volume (CV)to about 15×CV (e.g., between about 1×CV to about 14×CV, about 1×CV toabout 13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about2×CV to about 11×CV, about 3×CV to about 11×CV, about 2×CV to about5×CV, about 4×CV to about 11×CV, about 5×CV to about 11×CV, or about5×CV to about 10×CV).

In some of the processes described herein, the first MCCS includes areservoir that holds a fluid containing the recombinant therapeuticprotein at low pH (e.g., a pH below 4.6, below 4.4, below 4.2, below4.0, below 3.8, below 3.6, below 3.4, below 3.2, or below 3.0) for,e.g., about 1 minute to 1.5 hours (e.g., about 1 hour), and inactivatesthe viruses present in a fluid containing the recombinant therapeuticprotein. An example of a reservoir that can be used to perform the unitoperation of inactivating viruses is a stir flask (e.g., 500-mL stirflask, e.g., a 500-mL stir flask with a programmed stir plate) that iscapable of holding a fluid containing a recombinant therapeutic proteinfor, e.g., about 1 minute to 1.5 hours, before the fluid containing therecombinant therapeutic protein is fed into the second MCCS. Thereservoir that is used to perform the unit operation of inactivation ofviruses can be a 500-mL stir flask with a programmed stir plate (e.g., astir plate programmed to mix (e.g., periodically mix) the fluid withinthe reservoir, e.g., every 4 hours). Another example of a reservoir thatcan be used to perform the unit operation of inactivation of viruses isa plastic bag (e.g., 500-mL plastic bag) that is capable of holding afluid containing a recombinant therapeutic protein for, e.g., about 1minute to 1.5 hours, before the fluid containing the recombinanttherapeutic protein is fed into the second MCCS. In some examples, thefluid containing the recombinant therapeutic protein can already have alow pH (e.g., a pH below 4.6, below 4.4, below 4.2, below 4.0, below3.8, below 3.6, below 3.4, below 3.2, or below 3.0) when it is fed intothe reservoir that is used to perform the unit operation of viralinactivation. As can be appreciated by those skilled in the art, avariety of other means can be used to perform the unit operation ofinactivating viruses. For example, UV irradiation of a fluid containingrecombinant therapeutic protein can also be used to perform the unitoperation of inactivating viruses. Non-limiting examples of reservoirsthat can be used to perform the unit operation of inactivation ofviruses present in a fluid containing the recombinant therapeuticprotein are described herein.

The first MCCS can include a PCCS containing four chromatographycolumns, where at least three of the four chromatography columns performthe unit operation of capturing the recombinant therapeutic protein fromthe liquid culture medium (e.g., using a first MCCS that includes any ofthe at least one chromatography columns that contain a resin that iscapable of performing the unit operation of capturing (e.g., any ofthose described herein)). In these examples, the fourth-column of thePCC can perform the unit operation of inactivating viruses in a fluidthat contains the recombinant therapeutic protein (e.g., any of theexemplary columns described herein that can be used to achieve viralinactivation of a fluid containing the recombinant therapeutic protein).

A fluid containing the recombinant therapeutic protein is continuouslyeluted from the first MCCS (e.g., the first PCC system), and iscontinuously fed into the second MCCS. The percent of the recombinanttherapeutic protein recovered in the eluate of the first MCCS (e.g., thefirst PCC system) can be, e.g., at least 70%, at least 72%, at least74%, at least 76%, at least 78%, at least 80%, at least 82%, at least84%, at least 86%, at least 88%, at least 90%, at least 92%, at least94%, at least 96%, or at least 98%). The eluate from the first MCCS(e.g., the first PCC system) can be fed into the second MCCS (e.g.,second PCC system) using a variety of means known in the art (e.g.,tubing). The eluate of the first MCCS (e.g., first PCC system) can befed into the second MCCS (e.g., second PCC system) at a flow rate of,e.g., between about 0.2 mL/minute to about 25 mL/minute (e.g., betweenabout 0.2 mL/minute to about 20 mL/minute, between about 0.5 mL/minuteto about 20 mL/minute, between about 0.2 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 15 mL/minute, betweenabout 0.5 mL/minute to about 10 mL/minute, between about 0.5 mL/minuteand about 6.0 mL/minute, between about 1.0 mL/minute and about 5.0mg/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 5.0mL/minute to about 15.0 mL/minute, between about 15 mL/minute to about25 mL/minute, or between about 1.0 mL/minute and about 15.0 mL/minute).

Some processes described herein can further include a step of adjustingthe ionic concentration and/or pH of the eluate from the first MCCS(e.g., first PCC system) before it is fed into the second MCCS (e.g.,second PCC system). As described herein, the ionic concentration and/orpH of the eluate from the first MCCS (e.g., first PCC system) can beadjusted (before it is fed into the second MCCS) by adding a buffer tothe eluate (e.g., through the use of an in-line buffer adjustmentreservoir). The buffer can be added to the eluate from the first MCCS ata flow rate of, e.g., between about 0.1 mL/minute to about 15 mL/minute(e.g., between about 0.1 mL/minute to about 12.5 mL/minute, betweenabout 0.1 mL/minute to about 10.0 mL/minute, between about 0.1 mL/minuteto about 8.0 mL/minute, between about 0.1 mL/minute to about 6mL/minute, between about 0.1 mL/minute to 4 mL/minute, or between about0.5 mL/minute to about 5 mL/minute).

The processes described herein can further include a step of holding orstoring (and optionally also refridgerating) the eluate from the firstMCCS prior to feeding the eluate from the first MCCS into the secondMCCS. As described herein, this holding or storing step can be performedusing any of the reservoirs (e.g., back-up tanks) described herein.

The processes described herein can also include a step of filtering theeluate from the first MCCS before the eluate is fed into the secondMCCS. Any of the exemplary filters or methods for filtration describedherein can be used to filter the eluate from the first MCCS before theeluate is fed into the second MCCS.

Polishing and Purifying the Recombinant Therapeutic Protein

The processes described herein include a step of purifying and polishingthe recombinant therapeutic protein using a second MCCS, where theeluate from the MCC2 is a therapeutic protein drug substance. The secondMCCS can include at least one (e.g., two, three, or four) chromatographycolumn or chromatographic membrane that can be used to perform the unitoperation of purifying a recombinant therapeutic protein, and at leastone (e.g., two, three, or four) chromatography column or chromatographicmembrane that can be used to perform the unit operation of polishing therecombinant therapeutic protein.

The at least one chromatography column or chromatographic membrane thatcan be used to perform the unit operation of purifying the recombinanttherapeutic protein can contain a resin that utilizes a capturemechanism (e.g., any of the capture mechanisms described herein or knownin the art), or a resin that can be used to perform anion exchange,cation exchange, or molecular sieve chromatography. The at least onechromatography column or chromatographic membrane that can be used toperform the unit of operation of polishing the recombinant therapeuticprotein can contain a resin can be used to perform anion exchange,cation exchange, or molecular sieve chromatography (e.g., any of theexemplary resins for performing anion exchange, cation exchange, ormolecular sieve chromatography described herein or known in the art).

The size, shape, and volume of the at least one chromatography column orchromatography membrane that can be used to perform the unit ofoperation of purifying the recombinant therapeutic protein, and/or thesize and shape of the at least one chromatographic membrane that can beused to perform the unit of operation of polishing the recombinantmembrane can any of combination of the exemplary sizes, shapes, andvolumes of chromatography columns or chromatographic membranes describedherein. As can be appreciated by one skilled in the art, the step ofpurifying or polishing a recombinant therapeutic protein can, e.g.,include the steps of loading, washing, eluting, and equilibrating the atleast one chromatography column or chromatographic membrane used toperform the unit of operation of purifying or polishing the recombinanttherapeutic protein. Typically, the elution buffer coming out of achromatography column or chromatographic membrane used to perform theunit operation of purifying contains the recombinant therapeuticprotein. Typically, the loading and/or wash buffer coming out of achromatography column or chromatographic membrane used to perform theunit operation of polishing contains the recombinant therapeuticprotein.

For example, the size of the at least one chromatography column orchromatographic membrane that can be used to perform unit operation ofpurifying the recombinant therapeutic protein can have a volume of,e.g., between about 2.0 mL to about 200 mL (e.g., between about 2.0 mLto about 180 mL, between about 2.0 mL to about 160 mL, between about 2.0mL to about 140 mL, between about 2.0 mL to about 120 mL, between about2.0 mL to about 100 mL, between about 2.0 mL to about 80 mL, betweenabout 2.0 mL to about 60 mL, between about 2.0 mL to about 40 mL,between about 5.0 mL to about 40 mL, between about 2.0 mL to about 30mL, between about 5.0 mL to about 30 mL, or between about 2.0 mL toabout 25 mL). The flow rate of the fluid containing the recombinanttherapeutic protein as it is loaded onto the at least one chromatographycolumn or at least one chromatographic that can be used to perform theunit operation of purifying the recombinant therapeutic protein can be,e.g., between about 0.1 mL/minute to about 25 mL/minute (e.g., betweenabout 0.1 mL/minute to about 12.5 mL/minute, between about 0.1 mL/minuteto about 10.0 mL/minute, between about 0.1 mL/minute to about 8.0mL/minute, between about 0.1 mL/minute to about 6 mL/minute, betweenabout 0.1 mL/minute to 4 mL/minute, between about 0.1 mL/minute to about3 mL/minute, between about 0.1 mL/minute to about 2 mL/minute, or about0.2 mL/minute to about 4 mL/minute). The concentration of therecombinant therapeutic protein in the fluid loaded onto the at leastone chromatography column or chromatographic membrane that can be usedto perform the unit operation of purifying the recombinant therapeuticprotein can be, e.g., between about 0.05 mg/mL to about 100 mg/mLrecombinant protein (e.g., between about 0.1 mg/mL to about 90 mg/mL,between about 0.1 mg/mL to about 80 mg/mL, between about 0.1 mg/mL toabout 70 mg/mL, between about 0.1 mg/mL to about 60 mg/mL, between about0.1 mg/mL to about 50 mg/mL, between about 0.1 mg/mL to about 40 mg/mL,between about 0.1 mg/mL to about 30 mg/mL, between about 0.1 mg/mL toabout 20 mg/mL, between 0.5 mg/mL to about 20 mg/mL, between about 0.1mg/mL to about 15 mg/mL, between about 0.5 mg/mL to about 15 mg/mL,between about 0.1 mg/mL to about 10 mg/mL, or between about 0.5 mg/mL toabout 10 mg/mL recombinant therapeutic protein). The resin in the atleast one chromatography column or chromatographic membrane used toperform unit operation of purifying can be a resin that can be used toperform anion exchange or cation exchange chromatography. The resin inthe at least one chromatography column or chromatographic membrane thatis used to perform the unit operation of purifying can be a cationicexchange resin (e.g., Capto-S resin, GE Healthcare Life Sciences,Piscataway, N.J.).

Following the loading of the recombinant therapeutic protein onto the atleast one chromatographic column or chromatographic membrane in thesecond MCCS that can be used to perform the unit operation of purifyingthe recombinant therapeutic protein, the at least one chromatographiccolumn or chromatographic membrane is washed with at least one washingbuffer. As can be appreciated in the art, the at least one (e.g., two,three, or four) washing buffer is meant to elute all proteins that arenot the recombinant therapeutic protein from the at least onechromatography column or chromatographic membrane, while not disturbingthe interaction of the recombinant therapeutic protein with the resin orotherwise eluting the recombinant therapeutic protein.

The wash buffer can be passed through the at least one chromatographycolumn or chromatographic membrane at a flow rate of between about 0.2mL/minute to about 25 mL/minute (e.g., between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 1.0mL/minute and about 15.0 mL/minute). The volume of wash buffer used(e.g., combined total volume of wash buffer used when more than one washbuffer is used) can be, e.g., between about 1× column volume (CV) toabout 15×CV (e.g., between about 1×CV to about 14×CV, about 1×CV toabout 13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about2×CV to about 11×CV, about 3×CV to about 11×CV, about 4×CV to about11×CV, about 2.5×CV to about 5.0×CV, about 5×CV to about 11×CV, or about5×CV to about 10×CV). The total time of the washing can be, e.g.,between about 2 minutes to about 3 hours (e.g., between about 2 minutesto about 2.5 hours, between about 2 minutes to about 2.0 hours, betweenabout 5 minutes to about 1.5 hours, between about 10 minutes to about1.5 hours, between about 10 minutes to about 1.25 hours, between about20 minutes to about 1.25 hours, between about 30 minutes to about 1hour, between about 2 minutes and 10 minutes, between about 2 minutesand 15 minutes, or between about 2 minutes and 30 minutes).

Following the washing of the at least one chromatographic column orchromatographic membrane in the second MCCS that can be used to performthe unit operation of purifying the recombinant therapeutic protein, therecombinant therapeutic protein is eluted from the at least onechromatographic column or chromatographic membrane by passing an elutionbuffer through the at least one chromatographic column orchromatographic membrane in the second MCCS that can be used to performthe unit operation of purifying the recombinant therapeutic protein. Theelution buffer can be passed through the at least one chromatographycolumn or chromatographic membrane that can be used to perform the unitoperation of purifying the recombinant therapeutic protein at a flowrate of between about 0.2 mL/minute to about 25 mL/minute (e.g., betweenabout 0.2 mL/minute to about 20 mL/minute, between about 0.5 mL/minuteto about 20 mL/minute, between about 0.2 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 15 mL/minute, betweenabout 0.5 mL/minute to about 10 mL/minute, between about 0.5 mL/minuteand about 6.0 mL/minute, between about 1.0 mL/minute and about 5.0mg/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 1.0mL/minute and about 15.0 mL/minute). The volume of elution buffer usedto elute the recombinant therapeutic protein from each the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of purifying the recombinant therapeuticprotein can be, e.g., between about 1× column volume (CV) to about 25×CV(e.g., between about 1×CV to about 20×CV, between about 15×CV and about25×CV, between about 1×CV to about 14×CV, about 1×CV to about 13×CV,about 1×CV to about 12×CV, about 1×CV to about 11×CV, about 2×CV toabout 11×CV, about 3×CV to about 11×CV, about 4×CV to about 11×CV, about5×CV to about 11×CV, or about 5×CV to about 10×CV). The total time ofthe eluting can be, e.g., between about 2 minutes to about 3 hours(e.g., between about 2 minutes to about 2.5 hours, between about 2minutes to about 2.0 hours, between about 2 minutes to about 1.5 hours,between about 2 minutes to about 1.5 hours, between about 2 minutes toabout 1.25 hours, between about 2 minutes to about 1.25 hours, betweenabout 2 minutes to about 1 hour, between about 2 minutes and about 40minutes, between about 10 minutes and about 40 minutes, between about 20minutes and about 40 minutes, or between about 30 minutes and 1.0 hour).Non-limiting examples of elution buffers that can be used in thesemethods will depend on the resin and/or the therapeutic protein. Forexample, an elution buffer can contain a different concentration of salt(e.g., increased salt concentration), a different pH (e.g., an increasedor decreased salt concentration), or a molecule that will compete withthe recombinant therapeutic protein for binding to the resin. Examplesof such elution buffers for each of the exemplary capture mechanismsdescribed herein are well known in the art.

Following the elution of the recombinant therapeutic protein from the atleast one chromatographic column or chromatographic membrane in thesecond MCCS that can be used to perform the unit operation of purifyingthe recombinant therapeutic protein, and before the next volume of fluidcontaining a recombinant therapeutic protein can be loaded onto the atleast one chromatographic column or chromatographic membrane, the atleast one chromatography column or chromatographic membrane must beequilibrated using an regeneration buffer. The regeneration buffer canbe passed through the at least one chromatography column orchromatographic membrane that can be used to perform the unit operationof purifying the recombinant therapeutic protein at a flow rate of,e.g., between about 0.2 mL/minute to about 25 mL/minute (e.g., betweenabout 0.2 mL/minute to about 20 mL/minute, between about 0.5 mL/minuteto about 20 mL/minute, between about 0.2 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 15 mL/minute, betweenabout 0.5 mL/minute to about 10 mL/minute, between about 0.5 mL/minuteand about 6.0 mL/minute, between about 1.0 mL/minute and about 5.0mg/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 5.0mL/minute to about 15.0 mL/minute, or between about 1.0 mL/minute andabout 15.0 mL/minute). The volume of regeneration buffer used toequilibrate the at least one chromatography column or chromatographicmembrane that contains a resin that can be used to perform the unitoperation of purifying the recombinant therapeutic protein can be, e.g.,between about 1× column volume (CV) to about 15×CV (e.g., between about1×CV to about 14×CV, between about 1×CV to about 13×CV, between about1×CV to about 12×CV, between about 1×CV to about 11×CV, between about2×CV to about 11×CV, between about 3×CV to about 11×CV, between about2×CV to about 5×CV, between about 2.5×CV to about 7.5×CV, between about4×CV to about 11×CV, between about 5×CV to about 11×CV, or between about5×CV to about 10×CV). The concentration of recombinant therapeuticprotein in the eluate of the at least one chromatography column orchromatographic membrane that can be used to perform the unit operationof purifying the recombinant therapeutic protein can be, e.g., betweenabout 0.05 mg/mL to about 100 mg/mL recombinant therapeutic protein(e.g., between about 0.1 mg/mL to about 90 mg/mL, between about 0.1mg/mL to about 80 mg/mL, between about 0.1 mg/mL to about 70 mg/mL,between about 0.1 mg/mL to about 60 mg/mL, between about 0.1 mg/mL toabout 50 mg/mL, between about 0.1 mg/mL to about 40 mg/mL, between about2.5 mg/mL and about 7.5 mg/mL, between about 0.1 mg/mL to about 30mg/mL, between about 0.1 mg/mL to about 20 mg/mL, between 0.5 mg/mL toabout 20 mg/mL, between about 0.1 mg/mL to about 15 mg/mL, between about0.5 mg/mL to about 15 mg/mL, between about 0.1 mg/mL to about 10 mg/mL,or between about 0.5 mg/mL to about 10 mg/mL recombinant therapeuticprotein).

The at least one chromatography column or chromatographic membrane thatcan be used to perform the unit operation of polishing the recombinanttherapeutic protein can contain a resin that can be used to performcation exchange, anion exchange, or molecular sieve chromatography. Ascan be appreciated in the art, polishing a recombinant therapeuticprotein using the at least one chromatography column or chromatographymembrane that can be used to perform the unit operation of polishing therecombinant therapeutic protein in the second MCCS can include, e.g.,the steps of loading, chasing, and regenerating the at least onechromatography column or chromatographic membrane that can be used toperform the unit operation of polishing the recombinant therapeuticprotein. For example, when the steps of loading, chasing, andregenerating are used to perform the polishing, the recombinanttherapeutic protein does not bind the resin in the at least onechromatography column or chromatography membrane in the second MCCS thatis used to perform the unit operation of polishing the recombinanttherapeutic protein, and the recombinant therapeutic protein is elutedfrom the at least one chromatography column or chromatographic membranein the loading and chasing steps, and the regenerating step is used toremove any impurities from the at least one chromatography column orchromatographic membrane before additional fluid containing therecombinant therapeutic protein can be loaded onto the at least onechromatography column or chromatographic membrane. Exemplary flow ratesand buffer volumes to be used in each of the loading, chasing, andregenerating steps are described below.

The size, shape, and volume of the at least one chromatography column orchromatography membrane that can be used to perform the unit operationof polishing the recombinant therapeutic protein, and/or the size andshape of the at least one chromatographic membrane that can be used toperform the unit operation of polishing the recombinant therapeuticprotein can any of combination of the exemplary sizes, shapes, andvolumes of chromatography columns or chromatographic membranes describedherein. For example, the size of the at least one chromatography columnor chromatographic membrane that can be used to perform the unitoperation of polishing the recombinant therapeutic protein can have avolume of, e.g., between about 0.5 mL to about 200 mL (e.g., betweenabout 0.5 mL to about 180 mL, between about 0.5 mL to about 160 mL,between about 0.5 mL to about 140 mL, between about 0.5 mL to about 120mL, between about 0.5 mL to about 100 mL, between about 0.5 mL to about80 mL, between about 0.5 mL to about 60 mL, between about 0.5 mL toabout 40 mL, between about 5.0 mL to about 40 mL, between about 0.5 mLto about 30 mL, between about 5.0 mL to about 30 mL, between about 0.5mL to about 25 mL, between about 0.2 mL to about 10 mL, or between about0.2 mL to about 5 mL). The flow rate of the fluid containing therecombinant therapeutic protein as it is loaded onto the at least onechromatography column or chromatographic membrane that can be used toperform the unit operation of polishing the recombinant therapeuticprotein can be, e.g., between about 0.1 mL/minute to about 25 mL/minute(e.g., between about 0.1 mL/minute to about 12.5 mL/minute, betweenabout 0.1 mL/minute to about 10.0 mL/minute, between about 0.1 mL/minuteto about 8.0 mL/minute, between about 0.1 mL/minute to about 6mL/minute, between about 0.1 mL/minute to 4 mL/minute, between about 0.1mL/minute to about 3 mL/minute, between about 2 mL/minute and about 6mL/minute, between about 0.1 mL/minute to about 2 mL/minute, or about0.2 mL/minute to about 4 mL/minute). The total volume of fluidcontaining a recombinant therapeutic protein loaded onto the at leastone chromatography column or chromatographic membrane that can be usedto perform the unit operation of polishing the recombinant therapeuticprotein can be, e.g., between about 1.0 mL to about 250 mL (e.g.,between about 1.0 mL to about 225 mL, between about 1.0 mL to about 200mL, between about 1.0 mL to about 175 mL, between about 1.0 mL to about150 mL, between about 100 mL to about 125 mL, between about 100 mL toabout 150 mL, between about 1.0 mL to about 150 mL, between about 1.0 mLto about 125 mL, between about 1.0 mL to about 100 mL, between about 1.0mL to about 75 mL, between about 1.0 mL to about 50 mL, or between about1.0 mL to about 25 mL). The resin in the at least one chromatographycolumn or chromatographic membrane used to perform the polishing can bean anion exchange or cation exchange resin. The resin in the at leastone chromatography column or chromatographic membrane that is used toperform the unit operation of polishing can be a cationic exchange resin(e.g., Sartobind® Q resin, Sartorius, Goettingen, Germany).

Following the loading step, the at least one chromatographic column orchromatographic membrane in the second MCCS that can be used to performthe unit operation of polishing the recombinant therapeutic protein, achasing step is performed (e.g., a chase buffer is passed through the atleast one chromatography membrane or chromatographic membrane to collectthe recombinant therapeutic protein which does not substantially bind tothe at least one chromatography column or chromatographic membrane). Inthese examples, the chase buffer can be passed through the at least onechromatography column or chromatographic membrane at a flow rate ofbetween about 0.2 mL/minute to about 50 mL/minute (e.g., between about 1mL/minute to about 40 mL/minute, between about 1 mL/minute to about 30mL/minute, between about 5 mL/minute to about 45 mL/minute, betweenabout 10 mL/minute to about 40 mL/minute, between about 0.2 mL/minute toabout 20 mL/minute, between about 0.5 mL/minute to about 20 mL/minute,between about 0.2 mL/minute to about 15 mL/minute, between about 0.5mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 10mL/minute, between about 0.5 mL minute and about 14 mL/minute, betweenabout 1.0 mL/minute and about 25.0 mL/minute, between about 1.0mL/minute and about 15.0 mL/minute). The volume of chase buffer used canbe, e.g., between about 1× column volume (CV) to about 100×CV (e.g.,between about 1×CV to about 90×CV, between about 1×CV to about 80×CV,between about 1×CV to about 70×CV, between about 1×CV to about 60×CV,between about 1× to about 50×CV, between about 1×CV to about 40×CV,between about 1×CV to about 30×CV, between about 1×CV to about 20×CV,between about 1×CV to about 15×CV, between about 5×CV to about 20×CV,between about 5×CV to about 30×CV, between about 1×CV to about 14×CV,about 1×CV to about 13×CV, about 1×CV to about 12×CV, about 1×CV toabout 11×CV, about 2×CV to about 11×CV, about 3×CV to about 11×CV, about4×CV to about 11×CV, about 2.5×CV to about 5.0×CV, about 5×CV to about11×CV, or about 5×CV to about 10×CV). The total time of the chasing canbe, e.g., between about 1 minute to about 3 hours (e.g., between about 1minute to about 2.5 hours, between about 1 minute to about 2.0 hours,between about 1 minutes to about 1.5 hours, between about 2 minutes toabout 1.5 hours, between about 1 minutes to about 1.25 hours, betweenabout 2 minutes to about 1.25 hours, between about 1 minute to about 5minutes, between about 1 minute to about 10 minutes, between about 2minutes to about 4 minutes, between about 30 minutes to about 1 hour,between about 2 minutes and 10 minutes, between about 2 minutes and 15minutes, or between about 2 minutes and 30 minutes). The combinedconcentration of therapeutic recombinant protein present in the eluatecoming through the column in the loading step and the chasing step canbe, e.g., between about 0.1 mg/mL to about 100 mg/mL recombinant protein(e.g., between about 0.1 mg/mL to about 90 mg/mL, between about 0.1mg/mL to about 80 mg/mL, between about 0.1 mg/mL to about 70 mg/mL,between about 0.1 mg/mL to about 60 mg/mL, between about 0.1 mg/mL toabout 50 mg/mL, between about 0.1 mg/mL to about 40 mg/mL, between about2.5 mg/mL and about 7.5 mg/mL, between about 0.1 mg/mL to about 30mg/mL, between about 0.1 mg/mL to about 20 mg/mL, between 0.5 mg/mL toabout 20 mg/mL, between about 0.1 mg/mL to about 15 mg/mL, between about0.5 mg/mL to about 15 mg/mL, between about 0.1 mg/mL to about 10 mg/mL,between about 0.5 mg/mL to about 10 mg/mL, or between about 1 mg/mL andabout 5 mg/mL recombinant therapeutic protein).

Following the chasing step and before the next volume fluid containing arecombinant therapeutic protein can be loaded onto the at least onechromatographic column or chromatographic membrane that can be used toperform the unit operation of polishing, the at least one chromatographycolumn or chromatographic membrane must be regenerated using aregeneration buffer. The regeneration buffer can be passed through theat least one chromatography column or chromatographic membrane that canbe used to perform the unit operation of polishing the recombinanttherapeutic protein at a flow rate of, e.g., between about 0.2 mL/minuteto about 50 mL/minute (e.g., between about 1 mL/minute to about 40mL/minute, between about 1 mL/minute to about 30 mL/minute, betweenabout 5 mL/minute to about 45 mL/minute, between about 10 mL/minute toabout 40 mL/minute, between about 0.2 mL/minute to about 20 mL/minute,between about 0.5 mL/minute to about 20 mL/minute, between about 0.2mL/minute to about 15 mL/minute, between about 0.5 mL/minute to about 15mL/minute, between about 0.5 mL/minute to about 10 mL/minute, betweenabout 0.5 mL minute and about 14 mL/minute, between about 1.0 mL/minuteand about 25.0 mL/minute, between about 1.0 mL/minute and about 15.0mL/minute). The volume of regeneration buffer used to regenerate the atleast one chromatography column or chromatographic membrane that can beused to perform the unit operation of polishing can be, e.g., betweenabout 1× column volume (CV) to about 500×CV (e.g., between about 1×CV toabout 450×CV, between about 1×CV to about 400×CV, between about 1×CV toabout 350×CV, between about 1×CV to about 300×CV, between about 1×CV toabout 250×CV, between about 1×CV to about 200×CV, between about 1×CV toabout 150×CV, between about 1×CV to about 100×CV, between about 1×CV toabout 90×CV, between about 1×CV to about 80×CV, between about 1×CV toabout 70×CV, between about 1×CV to about 60×CV, between about 1× toabout 50×CV, between about 1×CV to about 40×CV, between about 1×CV toabout 30×CV, between about 1×CV to about 20×CV, between about 1×CV toabout 15×CV, between about 5×CV to about 20×CV, between about 5×CV toabout 30×CV, between about 1×CV to about 14×CV, about 1×CV to about13×CV, about 1×CV to about 12×CV, about 1×CV to about 11×CV, about 2×CVto about 11×CV, about 3×CV to about 11×CV, about 4×CV to about 11×CV,about 2.5×CV to about 5.0×CV, about 5×CV to about 11×CV, or about 5×CVto about 10×CV).

In other examples, the one or more chromatography column(s) and/orchromatographic membranes used to perform the unit operation ofpolishing contain a resin that selectively binds or retains theimpurities present in a fluid containing the recombinant therapeuticprotein, and instead of regenerating the one or more column(s) and/ormembrane(s), the one or more column(s) and/or membrane(s) are replaced(e.g., replaced with a substantially similar column(s) and/ormembrane(s)) once the binding capacity of the resin in the one or morecolumn(s) and/or membrane(s) has been reached or is substantially closeto being reached.

In some examples of these processes, the second MCCS includes a PCCScontaining three chromatography columns and one chromatographicmembrane, e.g., where the three chromatography columns in the PCCSperform the unit operation of purifying the recombinant therapeuticprotein (e.g., using at least one chromatography column(s) that can beused to perform the unit of operation of purifying the protein) and thechromatographic membrane in the PCCS performs the unit operation ofpolishing the recombinant therapeutic protein. In these examples, thechromatographic membrane in the PCCS that can be used to perform theunit operation of polishing the recombinant therapeutic protein can beany of the exemplary chromatographic membranes described herein that canbe used to perform the unit operation of polishing the recombinanttherapeutic protein. Any of the column switching methods describedherein can be used to determine when the first three chromatographycolumns and the chromatographic membrane in the PCCS in this example canbe switched.

Some embodiments of this example can further include a step of adjustingthe ionic concentration and/or pH of the eluate from the threechromatographic columns in the PCCS before the eluate is fed into thechromatographic membrane in the PCCS. As described herein, the ionicconcentration and/or pH of the eluate from the three chromatographycolumns in PCCS can be adjusted (before it is fed into thechromatographic membrane in the PCCS in this example)) by adding abuffer to the eluate of the three chromatography columns in the PCCS(e.g., through the use of an in-line buffer adjustment reservoir). Thebuffer can be added to the eluate at a flow rate of, e.g., between about0.1 mL/minute to about 15 mL/minute (e.g., between about 0.1 mL/minuteto about 12.5 mL/minute, between about 0.1 mL/minute to about 10.0mL/minute, between about 0.1 mL/minute to about 8.0 mL/minute, betweenabout 0.1 mL/minute to about 6 mL/minute, between about 0.1 mL/minute to4 mL/minute, or between about 0.5 mL/minute to about 5 mL/minute).

These examples can further include a step of holding or storing theeluate from the three chromatography columns in the PCCS in this exampleprior to feeding the eluate into the chromatographic membrane(chromatographic membrane that can be used to perform the unit operationof polishing the recombinant therapeutic protein). As described herein,this holding or storing step can be performed using any of thereservoirs (e.g., back-up tanks) described herein.

These examples can also include a step of filtering the eluate from thechromatographic membrane in the exemplary PCCS system (eluate of thechromatographic membrane that can be used to perform the unit operationof polishing the recombinant therapeutic protein). Any of the exemplaryfilters or methods for filtration described herein can be used to filterthe eluate from the chromatographic membrane in this exemplary PCCS(eluate of the chromatographic membrane that can be used to perform theunit operation of polishing the recombinant therapeutic protein).

As can be appreciated by those in the art, the therapeutic protein drugsubstance can be periodically eluted from the second MCCS using any ofthe processes described herein. For example, any of the processesdescribed herein can elute the therapeutic protein drug substance for aduration of, e.g., between about 30 seconds and about 5 hours (e.g.,between about 1 minute and about 4 hours, between about 1 minute andabout 3 hours, between about 1 minute and about 2 hours, between about 1minute or about 1.5 hours, between about 1 minute and about 1 hour,between about 1 minute and about 30 minutes) at a frequency of, e.g.,between about 1 minute and about 6 hours (e.g., between about 1 minuteand about 5 hours, between about 1 minute and about 4 hours, betweenabout 1 minute and about 3 hours, between about 1 minute and 2 hours,between about 1 minute and 1 hour, or between about 1 minute and 30minutes), depending on, e.g., the chromatography column(s) and/orchromatographic membrane(s) used in the first and second MCCS.

Culturing Methods

Some of the processes described herein further include a step ofculturing cells (e.g., recombinant mammalian cells) that secrete arecombinant therapeutic protein in a bioreactor (e.g., a perfusion orfed-batch bioreactor) that contains a liquid culture medium, wherein avolume of the liquid culture medium that is substantially free of cells(e.g., mammalian cells) is continuously or periodically removed from theperfusion bioreactor and fed into the first multi-column chromatographysystem (MCC1). The bioreactor can have a volume of, e.g., between about1 L to about 10,000 L (e.g., between about 1 L to about 50 L, betweenabout 50 L to about 500 L, between about 500 L to about 1000 L, between500 L to about 5000 L, between about 500 L to about 10,000 L, betweenabout 5000 L to about 10,000 L, between about 1 L and about 10,000 L,between about 1 L and about 8,000 L, between about 1 L and about 6,000L, between about 1 L and about 5,000 L, between about 100 L and about5,000 L, between about 10 L and about 100 L, between about 10 L andabout 4,000 L, between about 10 L and about 3,000 L, between about 10 Land about 2,000 L, or between about 10 L and about 1,000 L). The amountof liquid culture medium present in a bioreactor can be, e.g., betweenabout between about 0.5 L to about 5,000 L (e.g., between about 0.5 L toabout 25 L, between about 25 L to about 250 L, between about 250 L toabout 500 L, between 250 L to about 2500 L, between about 250 L to about5,000 L, between about 2500 L to about 5,000 L, between about 0.5 L andabout 5,000 L, between about 0.5 L and about 4,000 L, between about 0.5L and about 3,000 L, between about 0.5 L and about 2,500 L, betweenabout 50 L and about 2,500 L, between about 5 L and about 50 L, betweenabout 5 L and about 2,000 L, between about 5 L and about 1,500 L,between about 5 L and about 1,000 L, or between about 5 L and about 500L). Culturing cells can be performed, e.g., using a fed-batch bioreactoror a perfusion bioreactor. Non-limiting examples and different aspectsof culturing cells (e.g., culturing mammalian cells) are described belowand can be used in any combination.

Cells

The cells that are cultured in some of the processes described hereincan be bacteria (e.g., gram negative bacteria), yeast (e.g.,Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorphs,Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica, orArxula adeninivorans), or mammalian cells. The mammalian cell can be acell that grows in suspension or an adherent cell. Non-limiting examplesof mammalian cells that can be cultured in any of the processesdescribed herein include: Chinese hamster ovary (CHO) cells (e.g., CHODG44 cells or CHO-K1s cells), Sp2.0, myeloma cells (e.g., NS/0),B-cells, hybridoma cells, T-cells, human embryonic kidney (HEK) cells(e.g, HEK 293E and HEK 293F), African green monkey kidney epithelialcells (Vero) cells, and Madin-Darby Canine (Cocker Spaniel) kidneyepithelial cells (MDCK) cells. In some examples where an adherent cellis cultured, the culture can also contain a plurality of microcarriers(e.g., microcarriers that contain one or more pores). Additionalmammalian cells that can be cultured in any of the processes describedherein are known in the art.

The mammalian cell can contain a recombinant nucleic acid (e.g., anucleic acid stably integrated in the mammalian cell's genome) thatencodes a recombinant therapeutic protein. Non-limiting examples ofrecombinant nucleic acids that encode exemplary recombinant therapeuticproteins are described below, as are recombinant therapeutic proteinsthat can be produced using the methods described herein. In someinstances, the mammalian cell that is cultured in a bioreactor (e.g.,any of the bioreactors described herein) was derived from a largerculture.

A nucleic acid encoding a recombinant therapeutic protein can beintroduced into a mammalian cell using a wide variety of methods knownin molecular biology and molecular genetics. Non-limiting examplesinclude transfection (e.g., lipofection), transduction (e.g.,lentivirus, adenovirus, or retrovirus infection), and electroporation.In some instances, the nucleic acid that encodes a recombinanttherapeutic protein is not stably integrated into a chromosome of themammalian cell (transient transfection), while in others the nucleicacid is integrated. Alternatively or in addition, the nucleic acidencoding a recombinant therapeutic protein can be present in a plasmidand/or in a mammalian artificial chromosome (e.g., a human artificialchromosome). Alternatively or in addition, the nucleic acid can beintroduced into the cell using a viral vector (e.g., a lentivirus,retrovirus, or adenovirus vector). The nucleic acid can be operablylinked to a promoter sequence (e.g., a strong promoter, such as aβ-actin promoter and CMV promoter, or an inducible promoter). A vectorcontaining the nucleic acid can, if desired, also contain a selectablemarker (e.g., a gene that confers hygromycin, puromycin, or neomycinresistance to the mammalian cell).

In some instances, the recombinant therapeutic protein is a secretedprotein and is released by the mammalian cell into the extracellularmedium (e.g., the first and/or second liquid culture medium). Forexample, a nucleic acid sequence encoding a soluble recombinanttherapeutic protein can contain a sequence that encodes a secretionsignal peptide at the N- or C-terminus of the recombinant therapeuticprotein, which is cleaved by an enzyme present in the mammalian cell,and subsequently released into the extracellular medium (e.g., the firstand/or second liquid culture medium).

Culture Media

Liquid culture media are known in the art. The liquid culture media(e.g., a first and/or second tissue culture medium) can be supplementedwith a mammalian serum (e.g., fetal calf serum and bovine serum), and/ora growth hormone or growth factor (e.g., insulin, transferrin, andepidermal growth factor). Alternatively or in addition, the liquidculture media (e.g., a first and/or second liquid culture medium) can bea chemically-defined liquid culture medium, an animal-derived componentfree liquid culture medium, a serum-free liquid culture medium, or aserum-containing liquid culture medium. Non-limiting examples ofchemically-defined liquid culture media, animal-derived component freeliquid culture media, serum-free liquid culture media, andserum-containing liquid culture media are commercially available.

A liquid culture medium typically contains an energy source (e.g., acarbohydrate, such as glucose), essential amino acids (e.g., the basicset of twenty amino acids plus cysteine), vitamins and/or other organiccompounds required at low concentrations, free fatty acids, and/or traceelements. The liquid culture media (e.g., a first and/or second liquidculture medium) can, if desired, be supplemented with, e.g., a mammalianhormone or growth factor (e.g., insulin, transferrin, or epidermalgrowth factor), salts and buffers (e.g., calcium, magnesium, andphosphate salts), nucleosides and bases (e.g., adenosine, thymidine, andhypoxanthine), protein and tissue hydrolysates, and/or any combinationof these additives.

A wide variety of different liquid culture media that can be used toculture cells (e.g., mammalian cells) in any of the methods describedherein are known in the art. Medium components that also may be usefulin the present processes include, but are not limited to,chemically-defined (CD) hydrolysates, e.g., CD peptone, CD polypeptides(two or more amino acids), and CD growth factors. Additional examples ofliquid tissue culture medium and medium components are known in the art.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium described herein can be thesame type of media or different media.

Additional Features of Exemplary Bioreactors

The interior surface of any of the bioreactors described herein may haveat least one coating (e.g., at least one coating of gelatin, collagen,poly-L-ornithine, polystyrene, and laminin), and as is known in the art,one or more ports for the sparging of O₂, CO₂, and N₂ into the liquidculture medium, and a stir mechanism for agitating the liquid culturemedium. The bioreactor can incubate the cell culture in a controlledhumidified atmosphere (e.g., at a humidity of greater than 20%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or a humidity of 100%).The bioreactor can also be equipped with a mechanical device that iscapable of removing a volume of liquid culture medium from thebioreactor and optionally, a filter within the mechanical device thatremoves the cells from the liquid culture medium during the process oftransfer of the liquid culture medium out of the bioreactor (e.g., anATF system).

Temperature

The step of culturing of mammalian cells can be performed at atemperature of about 31° C. to about 40° C. Skilled practitioners willappreciate that the temperature can be changed at specific time point(s)in during the culturing step, e.g., on an hourly or daily basis. Forexample, the temperature can be changed or shifted (e.g., increased ordecreased) at about one day, two days, three days, four days, five days,six days, seven days, eight days, nine days, ten days, eleven days,twelve days, fourteen days, fifteen days, sixteen days, seventeen days,eighteen days, nineteen days, or about twenty days or more after theinitial seeding of the bioreactor with the cell (e.g., mammalian cell).For example, the temperature can be shifted upwards (e.g., a change ofup to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to or about 20degrees C.). For example, the temperature can be shifted downwards(e.g., a change of up to or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, orup to or about 20° C.).

CO₂

The culturing step described herein can further include exposing theliquid culture medium in the bioreactor to an atmosphere containing atmost or about 15% CO₂ (e.g., at most or about 14% CO₂, 12% CO₂, 10% CO₂,8% CO₂, 6% CO₂, 5% CO₂, 4% CO₂, 3% CO₂, 2% CO₂, or at most or about 1%CO₂).

Perfusion Bioreactor

The culturing step described herein can be performed using a perfusionbioreactor. Culturing a cell (e.g., a mammalian cell) in a perfusionbioreactor includes the removal from the bioreactor of a first volume ofa first liquid culture medium (e.g., containing any concentration ofmammalian cells, e.g., a first volume of a first liquid culture mediumthat is substantially free of cells), and adding to the first liquidculture medium a second volume of a second liquid culture medium.Removal and adding can be performed simultaneously or sequentially, or acombination of the two. Further, removal and adding can be performedcontinuously (e.g., at a rate that removes and replaces a volume ofbetween 0.1% to 800% (e.g., between 1% and 700%, between 1% and 600%,between 1% and 500%, between 1% and 400%, between 1% and 350%, between1% and 300%, between 1% and 250%, between 1% and 100%, between 100% and200%, between 5% and 150%, between 10% and 50%, between 15% and 40%,between 8% and 80%, and between 4% and 30%) of the volume of thebioreactor or the first liquid culture medium volume over any given timeperiod (e.g., over a 24-hour period, over an incremental time period ofabout 1 hour to about 24 hours, or over an incremental time period ofgreater than 24 hours)) or periodically (e.g., once every third day,once every other day, once a day, twice a day, three times a day, fourtimes a day, or five times a day), or any combination thereof. Whereperformed periodically, the volume that is removed or replaced (e.g.,within about a 24-hour period, within an incremental time period ofabout 1 hour to about 24 hours, or within an incremental time period ofgreater than 24 hours) can be, e.g., between 0.1% to 800% (e.g., between1% and 700%, between 1% and 600%, between 1% and 500%, between 1% and400%, between 1% and 300%, between 1% and 200%, between 1% and 100%,between 100% and 200%, between 5% and 150%, between 10% and 50%, between15% and 40%, between 8% and 80%, and between 4% and 30%) of the volumeof the bioreactor or the first liquid culture medium volume. The firstvolume of the first liquid culture medium removed and the second volumeof the second liquid culture medium added can in some instances be heldapproximately the same over each 24-hour period (or, alternatively, anincremental time period of about 1 hour to about 24 hours or anincremental time period of greater than 24 hours) over the entire orpart of the culturing period. As is known in the art, the rate at whichthe first volume of the first liquid culture medium is removed(volume/unit of time) and the rate at which the second volume of thesecond liquid culture medium is added (volume/unit of time) can bevaried. The rate at which the first volume of the first liquid culturemedium is removed (volume/unit of time) and the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be about the same or can be different.

Alternatively, the volume removed and added can change (e.g., graduallyincrease) over each 24-hour period (or alternatively, an incrementaltime period of between 1 hour and about 24 hours or an incremental timeperiod of greater than 24 hours) during the culturing period. Forexample the volume of the first liquid culture medium removed and thevolume of the second liquid culture medium added within each 24-hourperiod (or alternatively, an incremental time period of between about 1hour and above 24 hours or an incremental time period of greater than 24hours) over the culturing period can be increased (e.g., gradually orthrough staggered increments) over the culturing period from a volumethat is between 0.5% to about 20% of the bioreactor volume or the firstliquid culture medium volume to about 25% to about 150% of thebioreactor volume or the first liquid culture medium volume.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia. In other instances, the first liquid culture medium and thesecond liquid culture medium can be different.

The first volume of the first liquid culture medium can be removed,e.g., by a mechanical system that can remove the first volume of thefirst liquid culture medium from the bioreactor (e.g., the first volumeof the first liquid culture medium that is substantially free of cellsfrom the bioreactor). Alternatively or in addition, the first volume ofthe first liquid culture medium can be removed by seeping or gravityflow of the first volume of the first liquid culture medium through asterile membrane with a molecular weight cut-off that excludes the cell(e.g., mammalian cell).

The second volume of the second liquid culture medium can be added tothe first liquid culture medium in an automated fashion, e.g., byperfusion pump.

In some instances, removing the first volume of the first liquid culturemedium (e.g., a first volume of the first liquid culture medium that issubstantially free of mammalian cells) and adding to the first liquidculture medium a second volume of the second liquid culture medium doesnot occur within at least 1 hour (e.g., within 2 hours, within 3 hours,within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8hours, within 9 hours, within 10 hours, within 12 hours, within 14hours, within 16 hours, within 18 hours, within 24 hours, within 36hours, within 48 hours, within 72 hours, within 96 hours, or after 96hours) of the seeding of the bioreactor with a mammalian cell.

Fed-Batch Bioreactor

The culturing step described herein can be performed using a fed-batchbioreactor. Culturing a cell in a fed-batch bioreactor includes, overthe majority of the culturing period, the addition (e.g., periodic orcontinuous addition) to the first liquid culture medium of a secondvolume of a second liquid culture medium. The adding of the secondliquid culture medium can be performed continuously (e.g., at a ratethat adds a volume of between 0.1% to 300% (e.g., between 1% and 250%,between 1% and 100%, between 100% and 200%, between 5% and 150%, between10% and 50%, between 15% and 40%, between 8% and 80%, and between 4% and30%) of the volume of the bioreactor or the first liquid culture mediumvolume over any given time period (e.g., over a 24-hour period, over anincremental time period of about 1 hour to about 24 hours, or over anincremental time period of greater than 24 hours)) or periodically(e.g., once every third day, once every other day, once a day, twice aday, three times a day, four times a day, or five times a day), or anycombination thereof. Where performed periodically, the volume that isadded (e.g., within about a 24-hour period, within an incremental timeperiod of about 1 hour to about 24 hours, or within an incremental timeperiod of greater than 24 hours) can be, e.g., between 0.1% to 300%(e.g., between 1% and 200%, between 1% and 100%, between 100% and 200%,between 5% and 150%, between 10% and 50%, between 15% and 40%, between8% and 80%, and between 4% and 30%) of the volume of the bioreactor orthe first liquid culture medium volume. The second volume of the secondliquid culture medium added can in some instances be held approximatelythe same over each 24-hour period (or, alternatively, an incrementaltime period of about 1 hour to about 24 hours or an incremental timeperiod of greater than 24 hours) over the entire or part of theculturing period. As is known in the art, the rate at which the secondvolume of the second liquid culture medium is added (volume/unit oftime) can be varied over the entire or part of the culturing period. Forexample, the volume of the second liquid culture medium added can change(e.g., gradually increase) over each 24-hour period (or alternatively,an incremental time period of between 1 hour and about 24 hours or anincremental time period of greater than 24 hours) during the culturingperiod. For example the volume of the second liquid culture medium addedwithin each 24-hour period (or alternatively, an incremental time periodof between about 1 hour and above 24 hours or an incremental time periodof greater than 24 hours) over the culturing period can be increased(e.g., gradually or through staggered increments) over the culturingperiod from a volume that is between 0.5% to about 20% of the bioreactorvolume or the first liquid culture medium volume to about 25% to about150% of the bioreactor volume or the first liquid culture medium volume.The rate at which the second volume of the second liquid culture mediumis added (volume/unit of time) can be about the same over the entire orpart of the culturing period.

Skilled practitioners will appreciate that the first liquid culturemedium and the second liquid culture medium can be the same type ofmedia. In other instances, the first liquid culture medium and thesecond liquid culture medium can be different. The volume of the secondliquid culture medium can be added to the first liquid culture medium inan automated fashion, e.g., by perfusion pump.

In some instances, adding to the first liquid culture medium a secondvolume of the second liquid culture medium does not occur within atleast 1 hour (e.g., within 2 hours, within 3 hours, within 4 hours,within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9hours, within 10 hours, within 12 hours, within 14 hours, within 16hours, within 18 hours, within 24 hours, within 36 hours, within 48hours, within 72 hours, within 96 hours, or after 96 hours) of theseeding of the bioreactor with a mammalian cell. The cell culture mediumin fed-batch cultures is typically harvested at the end of cultureperiod and used in any of the processes described herein, however, thecell culture medium in fed-batch cultures can also be harvested at oneor more time points during the culturing period and used in any of theprocesses described herein.

Skilled practitioners will appreciate that any of the various cultureparameters (e.g., containers, volumes, rates or frequencies of replacingculture volumes, agitation frequencies, temperatures, media, and CO₂concentrations) can be used in any combination in to perform thesemethods. Further, any of the mammalian cells described herein or knownin the art can be used to produce a recombinant protein.

Exemplary Advantages

The processes described herein can result in a substantial increase inthe volumetric productivity of the recombinant therapeutic proteinpresent in the therapeutic protein drug substance. For example, theprocesses described herein can result at least a 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, and 10-fold increase in thevolumetric productivity of the recombinant therapeutic protein presentin the therapeutic protein drug substance. The biological activity of arecombinant therapeutic protein can be assessed using a variety ofmethods known in the art, and will depend on the activity of thespecific recombinant therapeutic protein. For example, the biologicalactivity of a recombinant therapeutic protein that is an immunoglobulin(e.g., an antibody or an antibody fragment) can be determined bymeasuring the affinity of the recombinant therapeutic antibody to bindto its specific epitope (e.g., using Biocore or competitiveenzyme-linked immunosorbent assays). The recombinant therapeutic proteinmay be an enzyme (e.g., a recombinant galactosidase, e.g., a recombinantalpha-galactosidase) and the biological activity may be determined bymeasuring the recombinant therapeutic enzyme's activity (e.g.,determining the catalytic rate constant of the recombinant therapeuticenzyme by measuring a decrease in the concentration of a detectablesubstrate or an increase in the concentration of a detectable product(e.g., using spectrophotometry or light emission). For example, thebiological activity of a recombinant therapeutic galactosidase can bedetected by measuring a decrease in the level of globotriasylceramide(GL-3) or galabiosylceramide, or an increase in the level of ceramidedihexoside or galactose.

The processes described herein can result in an increased percentage ofrecovery of the recombinant therapeutic protein (e.g., increasedpercentage of yield of the recombinant therapeutic protein present inthe liquid culture medium in the therapeutic protein drug substance).For example, the present processes can result in a percentage yield ofrecombinant therapeutic protein of greater than about 70%, greater thanabout 80%, greater than about 82%, greater than about 84%, greater thanabout 86%, greater than about 88%, greater than about 90%, greater thanabout 92%, greater than about 94%, greater than about 96%, or greaterthan about 98%. The present processes can result in a percentage yieldof between about 80% to about 90%, between about 82% to about 90%,between about 84% to about 90%, between about 84% to about 88%, betweenabout 84% to about 94%, between about 82% to about 92%, or between about85% to about 95%.

The concentration of recombinant therapeutic protein present in thetherapeutic protein drug substance can be greater than about 1.0 mg/mL,greater than about 1.5 mg/mL, greater than about 2.0 mg/mL, greater thanabout 2.5 mg/mL, greater than about 3.0 mg/mL, greater than about 3.5mg/mL, greater than about 4.0 mg/mL, greater than about 4.5 mg/mL,greater than about 5.0 mg/mL, greater than about 5.5 mg/mL, greater thanabout 6.0 mg/mL, greater than about 6.5 mg/mL, greater than about 7.0mg/mL, greater than about 7.5 mg/mL, greater than about 8.0 mg/mL,greater than about 8.5 mg/mL, greater than about 9.0 mg/mL, greater thanabout 10.0 mg/mL, greater than about 12.5 mg/mL, or greater than about15.0 mg/mL.

The processes described herein can result in a net yield of recombinanttherapeutic protein in the therapeutic protein drug substance of atleast about 5 g/day, at least about 6 g/day, at least about 7 g/day, atleast about 8 g/day, at least about 9 g/day, at least about 10 g/day, atleast about 11 g/day, at least about 12 g/day, at least about 13 g/day,at least about 14 g/day, at least about 15 g/day, at least about 16g/day, at least about 17 g/day, at least about 18 g/day, at least about19 g/day, at least about 20 g/day, at least about 25 g/day, at leastabout 30 g/day, at least about 35 g/day, or at least about 40 g/day overa continuous period of at least about 5 days, at least about 10 days, atleast about 15 days, at least about 20 days, at least about 25 days, atleast about 30 days, at least about 35 days, at least about 40 days, atleast about 45 days, at least about 50 days, at least about 55 days, atleast about 60 days, at least about 65 days, at least about 70 days, atleast about 75 days, at least about 80 days, at least about 85 days, atleast about 90 days, at least about 95 days, at least about 100 days, atleast about 110 days, at least about 120 days, at least about 130 days,at least about 140 days, at least about 150 days, at least about 160days, at least about 170 days, at least about 180 days, at least about190 days, at least about 200 days, at least about 210 days, at leastabout 220 days, at least about 230 days, at least about 240 days, atleast about 250 days, at least about 260 days, at least about 270 days,at least about 280 days, at least about 290 days, at least about 300days, at least about 310 days, at least about 320 days, at least about330 days, at least about 340 days, at least about 350 days, or at leastabout 365 days.

The processes provided herein can result in a significantly improvedspecific productivity rate. For example, the specific productivity rateachieved in the recombinant protein drug substance is at least 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold,170-fold, 180-fold, 190-fold, or 200-fold greater than the specificproductivity rate achieved using a different process (e.g., a batchpurification process or a process that is not integrated and/orcontinuous). The productivity in the recombinant protein drug substanceachieved by the present processes can be at least 10,000 units/L, atleast 15,000 units/L, at least about 20,000 units/L, at least about25,000 units/L, at least about 30,000 units/L, at least about 35,000units/L, or at least about 40,000 units/L (in the first and/or secondliquid culture medium). The productivity in the recombinant protein drugsubstance achieved by the present methods can be at least 1 g/L, atleast 1.5 g/L, at least 2.0 g/L, at least 2.5 g/L, at least 3.0 g/L, atleast 4.0 g/L, at least 4.5 g/L, or at least 5.0 g/L.

The processes described herein also provide for time-efficientproduction of a therapeutic drug substance from a liquid culture medium.For example, the elapsed time between feeding a fluid (e.g., a liquidculture medium) containing a therapeutic protein into the first MCCS andeluting a therapeutic protein drug substance (containing the therapeuticprotein) from the outlet of the second MCCS is, e.g., between about 4hours and about 48 hours, inclusive, e.g., between about 4 hours andabout 40 hours, between about 4 hours and about 35 hours, between about4 hours and about 30 hours, between about 4 hours and about 28 hours,between about 4 hours and about 26 hours, between about 4 hours andabout 24 hours, between about 4 hours and about 22 hours, between about4 hours and about 20 hours, between about 4 hours and about 18 hours,between about 4 hours and about 16 hours, between about 4 hours andabout 14 hours, between about 4 hours and about 12 hours, between about6 hours and about 12 hours, between about 8 hours and about 12 hours,between about 6 hours and about 20 hours, between about 6 hours andabout 18 hours, between about 6 hours and about 14 hours, between about8 hours and about 16 hours, between about 8 hours and about 14 hours,between about 8 hours and about 12 hours, between about 10 hours and 20hours, between about 10 hours and 18 hours, between about 10 hours and16 hours, between about 10 hours and 14 hours, between about 12 hoursand about 14 hours, between about 10 hours and about 40 hours, betweenabout 10 hours and about 35 hours, between about 10 hours and about 30hours, between about 10 hours and about 25 hours, between about 15 hoursand about 40 hours, between about 15 hours and about 35 hours, betweenabout 15 hours and about 30 hours, between about 20 hours and about 40hours, between about 20 hours and about 35 hours, or between about 20hours and about 30 hours, inclusive. In other examples, the elapsed timebetween feeding the fluid (e.g., a liquid culture medium) containing atherapeutic protein into the first MCCS and eluting a therapeuticprotein drug substance (containing the therapeutic protein) from theoutlet of the second MCCS is, e.g., greater than about 4 hours and isless than about 40 hours, inclusive, e.g., greater than 4 hours and lessthan about 39 hours, about 38 hours, about 37 hours, about 36 hours,about 35 hours, about 34 hours, about 33 hours, about 32 hours, about 31hours, about 30 hours, about 29 hours, about 28 hours, about 27 hours,about 26 hours, about 25 hours, about 24 hours, about 23 hours, about 22hours, about 21 hours, about 20 hours, about 19 hours, about 18 hours,about 17 hours, about 16 hours, about 15 hours, about 14 hours, about 13hours, about 12 hours, about 11 hours, about 10 hours, about 9 hours,about 8 hours, about 7 hours, about 6 hours, about 5 hours, or about 4.5hours, inclusive.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1 Use of a Single Periodic Counter-CurrentChromatography System (PCCS) in the Continuous Flow Processing of aRecombinant Therapeutic Protein

A set of initial experiments was performed in order to test parametersnecessary for the successful use of a single PCCS in the continuous flowprocessing of a recombinant therapeutic protein.

Materials and Methods

Cell Culture

Bioreactors with a working volume of 12 L (Broadley-James Corp., Irvine,Calif.) were operated in perfusion mode utilizing the ATF (RefineTechnologies) cell retention system with polyethersulfone 0.2-μmfilters. Sintered spargers (20-μm) were used for O₂ gas to maintain thedissolved O₂ set point, and drilled-hole spargers (990-μm) were used forN₂ gas to maintain the pCO₂ set point. Cell density in the cell culturewas monitored by offline measurements (Vi-CELL, Beckman Coulter, Brea,Calif.) and/or via online capacitance probes (Futura, Aber Instruments,Grand Island, N.Y.).

The bioreactor cell culture process runs utilized chemically-definedculture media and Chinese hamster ovary (CHO) cell lines that secreterecombinant therapeutic antibodies or recombinant therapeutic humanenzymes. The cell concentration in the bioreactor culture immediatelyfollowing inoculation was 0.5×10⁶ cells/mL. The cells were allowed togrow to 50-60×10⁶ cells/mL. Once the culture reached this cell density,cell-bleeding methods were initiated to maintain cell density at asteady state. Perfusion of the cell culture began at 24-h afterinoculation, at 1 reactor volume/day, with the rate of perfusionincreased proportional to the cell concentration in the culture. Asteady-state cell specific perfusion rate of 0.04-0.05 mL/cell-d wasmaintained. Dissolved O₂ in the bioreactor was kept above 30% of airsaturation. pH in the culture medium was maintained between 6.8 and 6.95through sodium carbonate addition. Antifoam (FoamAway, GIBCO, GrandIsland, N.Y.) was added to the liquid culture medium to control foamlevels. The liquid culture medium obtained from the bioreactors waspumped onto the single PCCS without additional clarification.

Periodic Counter-Current (PCC) Chromatographic System (PCCS)

The PCCS used in these experiments was a custom-modified ÄKTA (GEHealthcare, Piscataway, N.J.) system capable of running up to fourcolumns. The system was equipped with five UV monitors (UV-900), threepumps (P-900), multiple valves (PV-908, SV-903), one pH and oneconductivity meter (pH/C-900), and Unicorn-based custom software (GEHealthcare, Piscataway, N.J.).

Breakthrough Curves

Protein breakthrough curves are required to determine the appropriatecolumn switching strategy for the single PCCS. To obtain breakthroughcurves under the capture conditions, frontal loading experiments wereperformed. The dynamic binding capacity (DBC) was evaluated as afunction of residence time using clarified harvest and breakthroughprofiles measured by UV absorbance (280 nm). Column sizes of 6.0-mL and1.0-mL were used for the determination of DBC for the recombinanttherapeutic antibody and recombinant therapeutic human enzyme,respectively. The residence times were selected such that they weresufficiently long to satisfy the binding capacity requirements, whilealso ensuring that the loading time was longer than the rest of thecolumn operations (wash, elution, regeneration, etc.). Accordingly, theloading flow rate was adjusted to achieve a target residence time of 2.5minutes for the recombinant therapeutic antibody and 4.8 minutes for therecombinant therapeutic human enzyme.

Integration of the Single PCCS to the Bioreactor

In PCCSs, the residence time (RT) of the protein on the column can bedecreased without increasing the column size because the breakthroughfrom the first column in the system can be captured on the second columnin the system. This unique feature was used to design a continuousprocess such that the culture harvest could be processed at anyperfusion rate (D) by varying the column volume (V) and RT, as outlinedby Eq. 1:

V=D*RT  (1)

To achieve continuous capture of the recombinant protein, the singlePCCS was directly connected to the bioreactor as shown in FIG. 2. Theharvest from the bioreactor/ATF was pumped into a 2-L disposable bagserving as a small surge vessel (Hyclone, Logan, Utah) using aperistaltic pump (Masterflex, Cole-Parmer, Vernon Hills, Ill.). A 0.2-μmfilter (Millipack 40, Millipore, Billerica, Mass.) was added between thebioreactor and the surge bag as an additional sterile barrier. MabSelectSuRe (GE Healthcare, Piscataway, N.J.) and a Hydrophobic InteractionChromatographic (HIC) media in a XK16™, 1.6-cm×6-cm (GE Healthcare)column was used to capture the recombinant therapeutic antibody and therecombinant therapeutic human enzyme, respectively). The operation ofeach column consisted of equilibration, load, wash, elution, andregeneration steps. Since the engineering of the bench-scale single PCCSdid not allow for closed operation, sodium azide was added to theprocess stream in-line.

Analytical Methods

Recombinant Therapeutic Antibodies

In-house assays were used for the quantitation of the titer of host cellproteins (HCP), aggregation, residual protein A, and potency of therecombinant antibodies. Titer was measured using a Protein A column(Applied Biosystems, Carlsbad, Calif.). Residual protein A and HCP werequantitated by ELISA using antigen and antibodies produced in-house.Aggregation was measured by HPLC-SEC using a TSK-GEL, G3000SWXL, 7.8mM×30 cm, 5-μm column (TOSO HAAS, King of Prussia, Pa.). Recombinanttherapeutic antibody potency was measured by an in vitro cell-basedassay.

Recombinant Therapeutic Human Enzyme Activity Assay

The titer of recombinant therapeutic human enzyme in the column load andeluate was determined by measuring the hydrolysis rate of a syntheticsubstrate linked to p-nitrophenol (pNP) (Sigma Aldrich, St. Louis, Mo.).The samples (25-μL) were incubated with 225 μL of 40 μM substrate for 15minutes at 37° C. The reactions were quenched with 250 μL of 0.3 Mglycine, pH 10.5, and the absorbance was measured at 400 nm. One unit ofactivity was defined as the amount of recombinant therapeutic enzymerequired to hydrolyze one micromole of substrate to pNP per minute underthe defined assay conditions. Protein concentration was determined byRP-HPLC using a POROS R2/H 2.1×30 mM column (Applied Biosystems,Carlsbad, Calif.). The specific activity was expressed as pNP (units)/mgprotein

In-house assays were used for the quantitation of HCP, aggregation, andpurity. HCP was assayed by ELISA using proprietary reagents. Theaggregation (SEC-HPLC) assay used a TSK-GEL, G3000SWXL, 7.8 mM×30 cm,5-μm column (TOSO HAAS, King of Prussia, Pa.), while the RP-HPLC purityassay used a YMC Octyl 2 mm×100 mM, 5-μm column (Waters, Milford,Mass.).

Basic Concepts of the Single PCCS

A column operation generally consists of the load, wash, eluate, andregeneration steps. In the single PCCS, multiple columns are used to runthe same steps discretely and continuously in a cyclic fashion. Sincethe columns are operated in series, the flow through and wash from onecolumn is captured by a second column. This unique feature of PCCsystems allows for the loading of the resin close to its static bindingcapacity instead of to the dynamic binding capacity, as is typicalduring batch-mode chromatography. For the ease of illustration only, a3-column system is used to describe this principle of PCCS operation(FIG. 3). A cycle is defined as three complete column operationsresulting in three discrete elution pools. Once all the steps in a cycleare completed, the cycle is re-started. As a result, the feed stream isprocessed continuously in an operating PCC system, while recombinanttherapeutic protein elution from each column is discrete and periodic.

Column Switching Strategy

To advance from one step to another within a PCCS cycle (FIG. 3), acolumn switching strategy is employed. There are two automated switchingoperations required per column in the PCCS, the first of which isrelated to the initial recombinant therapeutic protein breakthrough,while the second coincides with column saturation. The single PCCSdescribed in this example was operated using a control strategyutilizing dynamic UV monitoring. In general, column switching can bedetermined by any Process Analytical Technology (PAT) tool capable ofin-line measurement of recombinant therapeutic protein concentrationwith feedback control. However, a PAT tool that operates in real-time,such as UV, is ideal for providing the trigger signal for columnswitching.

FIG. 4 illustrates the principle of column switching based on the UVabsorbance difference (ΔUV) between the feed inlet and column outlet.During column loading (Step 1; FIG. 3), the PCC control systemdetermines the impurity baseline when the absorbance stabilizes. As therecombinant therapeutic protein breaks through (Step 2; FIG. 3), thereis an increase in the outlet UV signal above the impurity baseline. Atthe point when ΔUV has reached a pre-determined threshold (such as 3%breakthrough of the recombinant therapeutic protein), the flow-throughfrom column 1 is directed onto column 2 instead of to the waste (t1;FIG. 4). When column 1 is nearly saturated with recombinant therapeuticprotein and the ΔUV has reached a pre-determined value (t2; FIG. 4), thefeed is switched to column 2. An important advantage of this ΔUV-basedcolumn switching strategy is that it allows for uniform loading of thecolumns irrespective of the feed recombinant therapeutic productconcentration and the column capacity. Within a reasonable range, thestrategy is adequate for harvest titer variability, thereby enhancingsystem robustness.

Accurate determination of the column-switching time, which is based onthe UV absorbance difference between the feed and column outlet, is oneof the critical elements of the single PCCS real-time control strategy.This requires synchronization of all five UV detectors (one feed andfour column outlet detectors) within a narrow range. The UV detectorswere calibrated using a 3% acetone solution. The detector path lengthswere manually adjusted so that all five absorbance values were within0.5% of one another. The path length adjustment was ≦10%.

Proof of Concept Using a Recombinant Antibody

Cell Culture

The model recombinant therapeutic antibody for this study was producedcontinuously over a 70-day period in a 12-L perfusion bioreactor underthe conditions described in Materials and Methods (FIG. 5). Thevolumetric productivity rate reached 1 g/L-d between days 30 and 40,then slowly declined for reasons that are yet to be determined. The peakvolumetric productivity rate was >5 fold higher than the fed-batchprocess using the same cell line, with a significant upward potentialfor the continuous process. It should be noted that the objective of thestudy was to demonstrate the functionality of the integrated continuoussystem. In fact, the volumetric production rate change allowed for thetesting of the robustness of the single PCCS and, particularly, itsability to handle variability in harvest titer.

Downstream

In batch chromatography, maximum DBC is achieved by increasing theresidence time (RT) and subsequently over-sizing the column. In PCCSs,the RT can be decreased without increasing the column size because thebreakthrough from first column can be captured on the second column.This advantage in PCCSs allows for smaller column sizes and shorter RT.In order for the single PCCS process to be continuous, RT has to belonger than the combined time taken by the rest of the process(equilibration, wash, elution, regeneration, etc.). Therefore, the sizeand number of columns to be used with a PCCS for a given process aredependent on the resin binding capacity, which dictates the length ofthe load step. The high-binding capacity of the MabSelect SuRe™ resin inthe recombinant antibody process (50 g/L) leads to a load step that islonger than the rest of the column steps combined, which allows forcontinuous recombinant therapeutic protein capture using a PCCScontaining three columns. Frontal loading experiments were utilized todetermine the breakthrough curves at different residence times for therecombinant antibody (data not shown), and a residence time of 2.5minutes was found to be optimal.

To test the long-term performance of the single PCCS, bioreactor harvestwas continuously captured for 30 days, which corresponds to 38 PCCcycles and 110 column operations, without any indications of time-basedperformance decline. The consistency of the continuous capture over thestudy duration was evaluated based on several performance indicators,such as chromatographic profile, recoveries, and recombinant antibodyCritical Quality Attributes (CQAs). The UV profile of the feed streamwas nearly constant over the duration of the run, and the 3 column UVoutlets were reproducible across various cycles. The recovery and fiveCQAs analyzed for the capture eluate were comparable between the 3columns, as well as over the entire period of continuous harvest capture(FIG. 6). These results demonstrated the feasibility of the directcontinuous recombinant antibody capture from a perfusion bioreactor,yielding consistent process performance indicators and CQAs over aprolonged period of time.

The single PCCS process was compared to an existing single column batchchromatography system in terms of the estimated chromatography columnfootprint and raw material consumption. Specifically, chromatographymedia capacity utilization was increased by 20%, buffer usage wasreduced by 25%, and individual column size was reduced 75 fold, in thesingle PCCS as compared to batch-mode purification.

Proof of Concept with Recombinant Human Enzyme

Cell Culture

The recombinant therapeutic human enzyme in this study was produced overa 70-day continuous cultivation in a 12-L bioreactor under theconditions described in Materials and Methods (FIG. 7). The volumetricproductivity rate reached 0.4 g/L-d around day 25, and remained steadyafter that with a CV of 16%, largely related to assay variability.Compared to the legacy process for the manufacture of the same molecule,this volumetric productivity is ˜40 fold higher, which is a result ofthe synergistic impact of the high cell density and the significantlyimproved cell specific production rate.

Downstream

The objective of this study was to determine the consistency of thecapture column performance indicators, CQAs of the capture eluate acrossthe columns and cycles, and the robustness of the single PCCS hardwareand control strategy over extended periods of time. Given that thebinding capacity of the HIC capture column was low (1 g/L resin), thetime required to load a column was shorter than the rest of the processsteps combined (wash, elution, regeneration, etc.). This resulted in asingle PCCS where 4 columns, instead of 3 columns, were required for theharvest to be processed continuously. Therefore, a single 4-column PCCSwas developed and used to continuously capture the recombinant humanenzyme.

The study was divided into two phases. The first phase consisted ofmethod development, where previously collected harvest was fed to thesingle PCCS from a sterile disposable bag. During the second phase, thesingle PCCS was directly integrated with the bioreactor for continuousprocessing, as outlined in FIG. 2. In the first phase of this study, thesingle PCCS was operated continuously for 9 days and 41 single PCCScycles or 164 column operations. The UV profile of the feed was constantover the entire run, and the 4 column outlet UV signals were reasonablyconsistent among the 4 columns and across the various cycles. Theconsistency of the single PCCS operation during the entire run wasdemonstrated by the analysis of additional column performance indicatorsand CQAs of the captured recombinant therapeutic protein (FIG. 8). Therecovery and the five CQAs analyzed were comparable between the fourcolumns over the period of 9 days of continuous harvest capture.

In the second phase of this study, recombinant therapeutic protein wasdirectly captured from a perfusion bioreactor by integrating the singlePCCS with the bioreactor, as shown in FIG. 2. The main objective was todemonstrate the performance of the continuous bioprocessing platformwith a highly complex, non-antibody protein over an extended period oftime. The column size and RT were scaled according to Equation 1 inorder to achieve continuous capture of the bioreactor harvest. Theintegrated single PCCS was operated continuously for 31 days and 160single PCCS cycles, which corresponds to 640 independent columnoperations, without any signs of time-based performance decline. Thefeed UV profile, the 4 column outlet UV profiles and CQAs wereconsistent for the entire duration of the PCCS operation. Since the datawere practically equivalent to those obtained in the first phase of thestudy, the time profiles are not shown.

In sum, these data indicate that a single PCCS can be used to processrecombinant therapeutic proteins (both recombinant therapeuticantibodies and recombinant therapeutic human enzymes) produced by amammalian cell culture.

Example 2 Use of a Two Periodic Counter-Current Chromatography Systems(PCCSs) in the Continuous Flow Processing of a Recombinant Protein

A system using two different PCCSs was generated which allows for thecontinuous bioprocessing of a recombinant therapeutic antibody from acell culture medium of a bioreactor. The eluate of the system containingthe recombinant therapeutic antibody is substantially ready forformulation as a pharmaceutical composition and is unformulatedtherapeutic protein drug substance.

Materials and Methods

Cell Culture

Bioreactors with a working volume of 10 L (Broadley-James Corp., Irvine,Calif.) were operated in perfusion mode utilizing the ATF (RefineTechnologies) cell retention system with polyethersulfone 0.2-μmfilters. Sintered spargers (20-μm) were used for O₂ gas to maintain thedissolved O₂ set-point, and drilled-hole spargers (990-μm) were used forN₂ gas to maintain the pCO₂ set-point in the culture medium. Celldensity in the bioreactor was monitored by offline measurements(Vi-CELL, Beckman Coulter, Brea, Calif.) and/or via online capacitanceprobes (Futura, Aber Instruments, Grand Island, N.Y.). The bioreactorcell culture process runs utilized chemically defined liquid culturemedia and a CHO cell line producing a recombinant therapeutic monoclonalantibody. After inoculation, the bioreactors contained in the liquidculture medium a viable cell concentration of 0.5×10⁶ cells/mL. Thecells were allowed to grow to a cell density of 30-40×10⁶ cells/mL. Oncethe cells reached this density, cell bleeding-methods were initiated tomaintain cell density at steady state. Perfusion began 24-h afterinoculation, and the volume of liquid culture medium removed andreplaced in the bioreactor increased proportionally with cellconcentration. A steady state cell specific perfusion rate of 0.04-0.05mL/cell-d was maintained. Dissolved O₂ was kept above 30% of airsaturation in the liquid culture medium. The pH of the liquid culturemedium was maintained between 6.8 and 6. 95 through sodium carbonateaddition. Antifoam (FoamAway, GIBCO, Grand Island, N.Y.) was used tocontrol foam levels in the bioreactor. The liquid culture mediumharvested from the bioreactors was pumped onto the two PCC systemwithout additional clarification.

Downstream

In order to develop an integrated and fully continuous process for theproduction of recombinant protein drug substance (DS) two PCCSs (PCCS1and PCCS2) were used (FIG. 9). Each PCCS performed multiple operationsin addition to allowing the use of periodic counter currentchromatography for continuous column operations. Since the engineeringof the bench-scale PCCS did not allow for closed operation, sodium azidewas added to the process stream in-line. This limitation of the smallscale system can be successfully addressed by proper design of largescale PCCS hardware.

Two Periodic Counter-Current (PCC) Chromatographic System

The two PCCS used in this study were a custom modified ÄKTA (GEHealthcare, Piscataway, N.J.) capable of running up to four columns.Each system was equipped with five UV monitors (UV-900), three pumps(P-900), multiple valves (PV-908, SV-903), one pH and one conductivitymeter (pH/C-900), and Unicorn-based custom software (GE Healthcare,Piscataway, N.J.).

Integration of Bioreactor to PCCS1 and PCCS2

The harvest from the bioreactor/ATF was pumped into a 2-L disposable bagserving as a small surge vessel (Hyclone, Logan, Utah) using aperistaltic pump (Masterflex, Cole-Parmer, Vernon Hills, Ill.). A 0.2-μmfilter (Millipack 40, Millipore, Billerica, Mass.) was added between thebioreactor and the surge bag as an additional sterile barrier. MabSelectSuRe (GE Healthcare, Piscataway, N.J.) Chromatographic media in a XK16™,1.6 cm×6 cm column was used to capture the recombinant therapeuticmonoclonal antibody. The eluate from each Protein A column (pH 3.75) waspumped into 250-mL glass, stirred reservoir with a residence time of 1hr to perform a viral inactivation (VI) operation. A 0.2 μm-filter(Millipack 100, Millipore, Billerica, Mass.) was added between PCCS1 andPCCS2 to act as a mixing device and a particulate barrier for downstreamcolumns.

Adjusted load material from PCCS1 was further purified on PCCS2, usingthree XK16™, 1.1 cm×7 cm (GE Healthcare) Capto S (GE Healthcare,Piscataway, N.J.) chromatography columns in a bind and elute mode.Finally Capto S eluate was polished using a 7-mL SartoBind Q-membrane(Sartorious XX), connected directly to Capto-S column outlets to yieldrecombinant protein drug substance. Additionally, in-line dilutionrequired prior to both loading on the Capto-S and SartoBind Q-membranewas performed using PCCS1 and PCCS2 pumps respectively (FIG. 9). Eachcolumn operation consisted of equilibration, load, wash, elution, andregeneration steps. The Sartobind Q-membrane consisted of equilibration,load, wash, and regeneration steps. Tables 1 and 2 show the processparameters for all the unit operations performed using PCCS1 and PCCS2,respectively. FIG. 9 also shows the flow rate for the liquids pumpedthrough the two PCCSs.

TABLE 1 Operational Parameters of the First PCCS (PCCS1) Stepdescription Parameter Value Capture by Column size (3 columns); proteinA 3.6 mL resin volume column Load Titer 0.6 mg/ml Load Flow rate 0.36CV/min Column residence time 2.8 min Total time of capture per 5.4 hours(325 min) column Load solo column 75 min Load on two columns in 135 min(1.5 L) series Elution flow rate 0.13 CV/min Elution volume 5 columnvolumes (CV) Elution time 40 min Wash 1 volume 4 CV wash 1 Time 19 minWash 2 volume 6 CV wash 2 time 28 min Regeneration volume 3 CVRegeneration Time 28 min protein Conc of the eluate ~8 mg/ml Δ UV cutoff 1 (t1) 3 percenrt Δ UV cut off 2 (t2) 70 percent percent recovery 75Low pH hold Time for hold 60 min Hold pH 3.75 Buffer adjustment Flowrate of Protein A 0.5 CV/min for S column load eluate transfer to PCC 2(In-line adjustment 1)

TABLE 2 Operational Parameters for the Second PCCS (PCCS2) Purifcationby Column size (3 columns); Capto-S column 6.7 mL resin volume Flow rateof transfer of 0.1 CV/min S-load Load Titer ~7 mg/ml Load Flow rate 0.1CV/min Load solo column 86 min Load on two columns in 22 min series Wash1 volume 2.5 CV wash 1 Time 5 min Wash 2 volume 2.5 CV wash 2 time 5 minElution flow rate Elution volume 17 CV Elution time 40 min Regenerationvolume 5 CV Regeneration Time 10 min protein Conc of the eluate ~4.5mg/mL Δ UV cut off 1 (t1) 3 percenrt Δ UV cut off 2 (t2) 70 percentpercent recovery 90 percenrt In-line buffer Elution flow rate of 0.07CV/min dilution adjustment buffer sartobind Q membrane volume 7 mlsmembrane polish (mem vol) step membrane equilibration 5 membranevolume/min flow rate Time of equilibration 10 min Load of Q-membraneflow 0.5 membrane vol/min   rate Load time Chase flow rate 5 membranevol/min Chase time 2 min Regeneration flow rate 5 membrane vol/minRegeneration time 10 min Pertcent Recovery 95 Protein concentration of~3.5 mg/mL the final therapeutic drug substance

Analytical Methods

In-house assays were used for the quantitation of titer host cellproteins (HCP), aggregation, residual protein A, and recombinantmonocloncal antibody potency. Titer was measured using a Protein Acolumn (Applied Biosystems, Carlsbad, Calif.). Residual protein A andHCP were quantitated by ELISA using antigen and antibodies producedin-house. Aggregation was measured by HPLC-SEC using a TSK-GEL,G3000SWXL, 7.8 mM×30 cm, 5-μm column (TOSO HAAS, King of Prussia, Pa.).Recombinant therapeutic monoclonal antibody potency was measured by anin vitro cell-based assay.

Column Switching Strategy

To advance from one step to another within a PCCS cycle (FIG. 3), acolumn switching strategy is employed. There are two automated switchingoperations required per column in each PCC, the first of which isrelated to the initial recombinant therapeutic protein breakthrough,while the second coincides with column saturation. The PCC systemdescribed in this work was operated using either a novel controlstrategy utilizing dynamic UV monitoring as well as time basedswitching. In general, column switching can be determined by any PATtool capable of in-line measurement of recombinant therapeutic proteinconcentration with feedback control

FIG. 4 illustrates the principle of column switching based on the UVabsorbance difference (ΔUV) between the feed inlet and column outlet.During column loading (Step 1; FIG. 3), the PCC control systemdetermines the impurity baseline when the absorbance stabilizes. As therecombinant therapeutic protein breaks through (Step 2; FIG. 3), thereis an increase in the outlet UV signal above the impurity baseline. Atthe point when ΔUV has reached a pre-determined threshold (such as 3%breakthrough of the recombinant therapeutic protein), the flow-throughfrom column 1 is directed onto column 2 instead of to the waste (t1;FIG. 4). When column 1 is nearly saturated with recombinant therapeuticprotein and the ΔUV has reached a pre-determined value (t2; FIG. 4), thefeed is switched to column 2. An important advantage of this ΔUV-basedcolumn switching strategy is that it allows for uniform loading of thecolumns irrespective of the feed recombinant therapeutic proteinconcentration and the column capacity. Within a reasonable range, thestrategy is adequate for harvest titer variability, thereby enhancingsystem robustness.

As discussed above, accurate determination of the column-switching time,which is based on the UV absorbance difference between the feed andcolumn outlet, is one of the critical elements of the PCC real-timecontrol strategy. This requires synchronization of all five UV detectors(one feed and four column outlet detectors) within a narrow range. TheUV detectors were calibrated using a 3% acetone solution. The detectorpath lengths were manually adjusted so that all five absorbance valueswere within 0.5% of one another. The path length adjustment was ≦10%.

The second strategy used for column switching was with respect to time.Since the recombinant therapeutic protein titer over 31 days ofproduction was quasi-steady state, the column switching could becalculated based on time it took for pre-determined threshold as well assaturation.

Results and Discussion

To test the long-term performance of the two PCC system, the liquidculture medium harvested from the bioreactor was continuously purifiedfor 31 days, which corresponds to 372 purification cycles, or 25 two PCCsystem batches of production, without any indications of time-basedperformance decline. The consistency of the continuous capture over thestudy duration was evaluated based on several performance indicators,such as chromatographic profile, recoveries, and recombinant therapeuticmonoclonal antibody Critical Quality Attributes (CQAs). The UV profileof the feed stream was nearly constant over the duration of the run, andthe 3 column UV outlets were reproducible across various cycles. Thesedetails and the cycle-to-cycle reproducibility are demonstrated in therun chromatogram snapshot shown in FIGS. 12-14. The recovery and fiveCQAs analyzed for the capture eluate were comparable between the 3columns, as well as over the entire period of continuous harvest capture(FIG. 16). These results demonstrate the feasibility of the directcontinuous recombinant therapeutic monoclonal antibody capture from aperfusion bioreactor using a two PCC system, yielding consistent processperformance indicators and CQAs over a prolonged period of time. A modelrecombinant therapeutic monoclonal antibody was used to study thefeasibility of fully continuous bioprocessing platform using anintegrated two PCC system.

Proof of Concept Using a MAb

Cell Culture

The model recombinant therapeutic monoclonal antibody for this study wasproduced continuously over a 59-day period in a 12-L perfusionbioreactor under the conditions described in Materials and Methods. Thevolumetric productivity was maintained between 1.0-1.6 g/L from day 10through day 59. The peak volumetric productivity was ˜8 fold higher thanthe fed batch process using the same cell line, with a significantupward potential for the continuous process by optimizing perfusion rateand/or increasing the steady state cell density (FIGS. 11 and 12). Itshould be noted that the objective of the study was to demonstrate thefunctionality of the integrated continuous system. In fact, thevolumetric production rate change allowed for the testing of therobustness of the PCC systems and, particularly, its ability to handlevariability in harvest titer.

Downstream

The main objective was to demonstrate the performance of the continuousbioprocessing platform with a recombinant therapeutic monoclonalantibody using an integrated two PCC system over an extended period oftime. The column/membrane size and residence time for both the Protein Aand the cation exchange column were scaled to achieve continuousprocessing of the bioreactor harvest to unformulated drug substance(DS). The integrated system was operated continuously for 31 days whichcorresponds to 15 independent unformulated DS batches (batch is definedas 1.25 days of processing, 15 Q-membrane filtrates or 0.8 g of DS),without any signs of time-based performance decline. The feed UVprofile, the 3-column outlet UV profiles for protein A capture andCapto-S, as well as Sartobind Q membrane filtrates, were constant forthe entire duration of the integrated bioreactor-PCC1-PCC2 operation(FIGS. 12-14). Additionally, all CQAs for unformulated DS wereconsistent for the entire duration of the PCC operation (FIGS. 15 and16). The advantages of using the integrated continuous two PCC systemfor production of recombinant therapeutic monoclonal antibody ascompared to the existing batch purification processes include anincrease in the volumetric productivity, an increase in thechromatography media capacity utilization, a decrease in the volume ofbuffer usage, and a decrease in the volume of the individual columnsize.

Implementation of the Fully Continuous Biomanufacturing Platform

Currently, there are two dominant platforms for biopharmaceuticalmanufacturing: (1) perfusion bioreactors, and (2) fed-batch bioreactorsfor production of stable proteins. In both cases, non-limiting exemplarysystems described herein provide for a bioreactor operation followed bymultiple batch unit operations, including clarification, capture,polishing chromatography, and hold steps. The exemplary fully continuousproduction technology described in this Example allows for streamlinedrecombinant therapeutic protein production. As demonstrated in thisExample, this exemplary system in combination with high producing clonesand chemically-defined media can achieve very high cell density andvolumetric productivity while operating at steady state. As a result,sufficient production capacity can be achieved with smaller bioreactors(<500 L) versus traditional processes where reactor scales exceed 10,000L. The use of the ATF cell separation device eliminates theclarification unit operation. Most importantly, the direct integrationof the fully continuous production makes harvest hold tanks obsolete,and replaces the large batch capture column with up to 2orders-of-magnitude smaller columns used in the continuous system.Furthermore, continuous processing of the harvested liquid culturemedium confers significant advantages with respect to recombinanttherapeutic protein quality. Specifically, elimination of the harvestand other hold steps decreases target recombinant therapeutic proteinexposure to enzymatic, chemical, and physical degradation, and therebymitigates recombinant therapeutic protein stability risks. In summary,the successful development of the fully continuous bioprocessingplatform at small scale for the manufacture of recombinant monoclonalantibodies opens up the potential for its large scale industrialimplementation. These data show that the exemplary integrated,fully-continuous bioprocessing system offers unique advantages overtraditional approaches for recombinant therapeutic proteinmanufacturing. For example, Tables 3 and 4 below list a few of theadvantages provided by the exemplary process and system described inthis Example.

TABLE 3 Exemplary Advantages of Two PCC System Continous ProcessingProtein A Capto-S Parameters Units Batch column column Resin capacityNormalized (%) 100 120  110  Buffer Usage Normalized (%) 100 75 80Column Volume Normalized (%) 100 3.9 (X 3) 4.7 (X3) Diameter Normalized(%) 100 16 40 Height Normalized (%) 100 47 30

TABLE 4 Exemplary Advantages of Two PCC System Continuous PlatformParameters Current Batch Process Upstream Cycle Time 1 BRX volume/ 1 BRXvolume/ 14 days 12 hours Downstream Cycle Time Days-Months ~10 hoursVolumetric Productivity ~0.1-0.2 ~1.2 (g/L-Day) Automation Batch mode,discrete Fully automatic unit ops Total Number of 9 (harvest,Centrifugation, 3 (PCC1, PCC2 downstream unit depth filtration, Pro A,VI, and Viral Operations Capto S, Capto Q, viral filtration) filtration,sterile filtration) Hold Steps 9 None

Example 3 Exemplary Two-PCC System that Includes a 500-L Bioreactor

The method and system described below can be used to perform continuousbioprocessing of a recombinant therapeutic protein harvested from a500-L bioreactor culture.

Materials and Methods

Cell Culture

Bioreactors with a working volume of 500-L are operated in perfusionmode utilizing the ATF (Refine Technologies) cell retention system withpolyethersulfone 0.2-μm filters. Sintered spargers (20-μm) are used forO₂ gas to maintain the dissolved O₂ set point, and drilled-hole spargers(990-μm) are used for N₂ gas to maintain the pCO₂ set point. Celldensity in the cell culture is monitored by offline measurements(Vi-CELL, Beckman Coulter, Brea, Calif.) and/or via online capacitanceprobes (Futura, Aber Instruments, Grand Island, N.Y.).

The bioreactor cell culture process runs utilize chemically-definedculture media and Chinese hamster ovary (CHO) cell lines that secreterecombinant antibodies or recombinant human enzymes. The cellconcentration in the bioreactor culture immediately followinginoculation is 0.5×10⁶ cells/mL. The cells are allowed to grow to50-60×10⁶ cells/mL. Once the culture reach this cell density,cell-bleeding methods are initiated to maintain cell density at a steadystate. Perfusion of the cell culture begins at 24-h after inoculation,at 1 reactor volume/day, with the rate of perfusion increasedproportional to the cell concentration in the culture. A steady-statecell specific perfusion rate of 0.04-0.05 mL/cell-d is maintained.Dissolved O₂ in the bioreactor is kept above 30% of air saturation. pHin the culture medium is maintained between 6.8 and 6.95 through sodiumcarbonate addition. Antifoam (FoamAway, GIBCO, Grand Island, N.Y.) isadded to the liquid culture medium to control foam levels. The liquidculture medium obtained from the bioreactors is pumped onto the singlePCC system without additional clarification.

Periodic Counter-Current (PCC) Chromatographic Systems

The PCC systems to be used in these experiments are custom-modified ÄKTA(GE Healthcare, Piscataway, N.J.) system capable of running up to fourcolumns. The system is equipped with five UV monitors (UV-900), threepumps (P-900), multiple valves (PV-908, SV-903), one pH and oneconductivity meter (pH/C-900), and Unicorn-based custom software (GEHealthcare, Piscataway, N.J.). The first PCCS is a four-column PCCS,where the first three columns contain a protein A binding resin andperform the unit operation of capturing the recombinant therapeuticprotein from a fluid, and the fourth column performs the unit operationof inactivating viruses present in a fluid (e.g., capable of holding thefluid for about 1 hours at a pH of 3.75). The second PCCS contains atotal of three chromatography columns that perform the unit operation ofpurifying the recombinant therapeutic protein (containing a cationicexchange resin) and one chromatographic membrane that performs the unitoperation of polishing (containing a cationic exchange resin). Thespecific flow rates and other properties of the first and second PCCsystems to be used to continuously produce a recombinant protein drugsubstance are shown in Tables 5 and 6, respectively.

TABLE 5 Operational Parameters for the First PCCS Step descriptionParameter Value Capture by Column size (3 columns); protein A 1.6 Lresin volume column Load Titer 0.6 mg/mL Load Flow rate 0.36 CV/minColumn residence time 2.8 min Total time of capture per 5.4 hours (325min) column Load solo column 75 min Load on two columns in 135 min (1.5L) series Elution flow rate 0.13 CV/min Elution volume 5 column volumes(CV) Elution time 40 min Wash 1 volume 4 CV wash 1 Time 19 min Wash 2volume 6 CV wash 2 time 28 min Regeneration volume 3 CV RegenerationTime 28 min protein Conc of the eluate ~8 mg/mL Δ UV cut off 1 (t1) 3percenrt Δ UV cut off 2 (t2) 70 percent percent recovery 75 Low pH holdTime for hold 60 min Buffer adjustment Hold pH 3.75 for S column Flowrate of Protein A 0.5 CV/min load (In-line eluate transfer to PCC 2adjustment 1)

TABLE 6 Operational Parameters for the Second PCCS Purifcation Columnsize (3 columns); by Capto-S 335 mL resin volume column Flow rate oftransfer of 0.1 CV/min S-load Load Titer ~7 mg/mL Load Flow rate 0.1CV/min Load solo column 86 min Load on two columns in 22 min series Wash1 volume 2.5 CV wash 1 Time 5 min Wash 2 volume 2.5 CV wash 2 time 5 minElution flow rate Elution volume 17 CV Elution time 40 min Regenerationvolume 5 CV Regeneration Time 10 min protein Conc of the eluate ~4.5mg/mL Δ UV cut off 1 (t1) 3 percenrt ΔUV cut off 2 (t2) 70 percentpercent recovery 90 percenrt In-line buffer Elution flow rate of 0.07CV/min dilution adjustment buffer sartobind Q membrane volume 350 mlsmembrane membrane equilibration 5 membrane vol/min polish step flow rateTime of equilibration 10 min Load of Q-membrane flow 0.5 membranevol/min   rate Load time Chase flow rate 5 membrane vol/min Chase time 2min Regeneration flow rate 5 membrane vol/min Regeneration time 10 minPertcent Recovery 95 Protein concentration of ~3.5 mg/mL the finaltherapeutic drug substance

Breakthrough Curves

Protein breakthrough curves are required to determine the appropriatecolumn switching strategy for each PCC system. To obtain breakthroughcurves under the capture conditions, frontal loading experiments areperformed. The dynamic binding capacity (DBC) is evaluated as a functionof residence time using clarified harvest and breakthrough profilesmeasured by UV absorbance (280 nm). Column sizes of 6.0-mL and 1.0-mLare used for the determination of DBC for recombinant therapeuticantibodies and recombinant therapeutic human enzyme, respectively. Theresidence times are selected such that they are sufficiently long tosatisfy the binding capacity requirements, while also ensuring that theloading time is longer than the rest of the column operations (wash,elution, regeneration, etc.).

Integration of the Single PCC System to the Bioreactor

In PCC systems, the residence time (RT) of the protein on the column canbe decreased without increasing the column size because the breakthroughfrom the first column in the system can be captured on the second columnin the system. This unique feature is used to design a continuousprocess such that the culture harvest could be processed at anyperfusion rate (D) by varying the column volume (V) and RT, as outlinedby Eq. 1:

V=D*RT  (1)

To achieve continuous capture of the recombinant protein, the firstsingle PCCS is directly connected to the bioreactor as shown in FIG. 2.The harvest from the bioreactor/ATF is pumped into a 2-L disposable bagserving as a small surge vessel (Hyclone, Logan, Utah) using aperistaltic pump (Masterflex, Cole-Parmer, Vernon Hills, Ill.). A 0.2-μmfilter (Millipack 40, Millipore, Billerica, Mass.) is added between thebioreactor and the surge bag as an additional sterile barrier. MabSelectSuRe (GE Healthcare, Piscataway, N.J.) column is used to capture therecombinant therapeutic antibody and the recombinant therapeutic humanenzyme, respectively). The operation of each column consists ofequilibration, load, wash, elution, and regeneration steps. Since theengineering of the bench-scale single PCC system does not allow forclosed operation, sodium azide is added to the process stream in-line.

Analytical Methods

Recombinant Antibodies

In-house assays are used for the quantitation of the titer of host cellproteins (HCP), aggregation, residual protein A, and potency of therecombinant antibodies. Titer is measured using a Protein A column(Applied Biosystems, Carlsbad, Calif.). Residual protein A and HCP arequantitated by ELISA using antigen and antibodies produced in-house.Aggregation is measured by HPLC-SEC using a TSK-GEL, G3000SWXL, 7.8mM×30 cm, 5-μm column (TOSO HAAS, King of Prussia, Pa.). Recombinantantibody potency is measured by an in vitro cell-based assay.

Basic Concepts of the Single PCC System

A column operation generally consists of the load, wash, eluate, andregeneration steps. In each PCC system, multiple columns will be used torun the same steps discretely and continuously in a cyclic fashion.Since the columns are operated in series, the flow through and wash fromone column is captured by a second column. This unique feature of PCCsystems allows for the loading of the resin close to its static bindingcapacity instead of to the dynamic binding capacity, as is typicalduring batch-mode chromatography. For the ease of illustration, a3-column system is used to describe this principle of PCC systemoperation (FIG. 3). A cycle is defined as three complete columnoperations resulting in three discrete elution pools. Once all the stepsin a cycle are completed, the cycle is re-started. As a result, the feedstream is processed continuously in an operating PCC system, whilerecombinant therapeutic protein elution from each column is discrete andperiodic.

Column Switching Strategy

To advance from one step to another within a PCC system cycle (FIG. 3),a column switching strategy is employed. There are two automatedswitching operations required per column in the PCC system, the first ofwhich is related to the initial recombinant therapeutic proteinbreakthrough, while the second coincides with column saturation. Eachsingle PCC system described in this example is operated using a controlstrategy utilizing dynamic UV monitoring. In general, column switchingcan be determined by any Process Analytical Technology (PAT) toolcapable of in-line measurement of recombinant therapeutic proteinconcentration with feedback control. However, a PAT tool that operatesin real time, such as UV, is ideal for providing the trigger signal forcolumn switching.

FIG. 4 illustrates the principle of column switching based on the UVabsorbance difference (ΔUV) between the feed inlet and column outlet.During column loading (Step 1; FIG. 3), the PCC control systemdetermines the impurity baseline when the absorbance stabilizes. As therecombinant therapeutic protein breaks through (Step 2; FIG. 3), thereis an increase in the outlet UV signal above the impurity baseline. Atthe point when ΔUV has reached a pre-determined threshold (such as 3%breakthrough of the recombinant therapeutic protein), the flow-throughfrom column 1 is directed onto column 2 instead of to the waste (t1;FIG. 4). When column 1 is nearly saturated with recombinant therapeuticprotein and the ΔUV has reached a pre-determined value (t2; FIG. 4), thefeed is switched to column 2. An important advantage of this ΔUV-basedcolumn switching strategy is that it allows for uniform loading of thecolumns irrespective of the feed recombinant therapeutic proteinconcentration and the column capacity. Within a reasonable range, thestrategy is adequate for harvest titer variability, thereby enhancingsystem robustness.

Accurate determination of the column-switching time, which is based onthe UV absorbance difference between the feed and column outlet, is oneof the critical elements of the each single PCC system real-time controlstrategy. This requires synchronization of all five UV detectors (onefeed and four column outlet detectors) within a narrow range. The UVdetectors are calibrated using a 3% acetone solution. The detector pathlengths are manually adjusted so that all five absorbance values arewithin 0.5% of one another. The path length adjustment is ≦10%.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An integrated and continuous process for manufacturing a therapeuticprotein drug substance, the process comprising: (i) providing a liquidculture medium comprising a recombinant therapeutic protein that issubstantially free of cells, wherein the liquid culture medium is fedinto a first multi-column chromatography system (MCCS1); (ii) capturingsaid recombinant therapeutic protein in the liquid culture medium usingthe MCCS1, wherein the eluate of the MCCS1 containing the recombinanttherapeutic protein is continuously fed into a second multi-columnchromatography system (MCCS2); and (iii) purifying and polishing therecombinant therapeutic protein using the MCCS2, wherein the eluate fromthe MCCS2 is a therapeutic protein drug substance; and wherein theprocess is integrated and runs continuously from said liquid culturemedium to the eluate from the MCCS2 that is the therapeutic protein drugsubstance.
 2. (canceled)
 3. The process of claim 1, wherein the MCCS1and/or the MCCS2 performs at least two different unit operations.
 4. Theprocess of claim 1, wherein the use of the MCCS1 or the MCCS2, or both,involves column switching.
 5. The process of claim 3, wherein the MCCS1performs the unit operations of capturing the recombinant therapeuticprotein and inactivating viruses.
 6. The process of claim 3, wherein theMCCS2 performs the unit operations of purifying and polishing therecombinant therapeutic protein. 7.-10. (canceled)
 11. The process ofclaim 1, wherein the MCCS1 is a first periodic counter currentchromatography system (PCCS1).
 12. The process of claim 11, where thePCCS1 comprises a four-column PCCS.
 13. The process of claim 12, whereinthree of the four columns in the four-column PCCS perform the unitoperation of capturing the recombinant therapeutic protein from theliquid culture medium. 14.-16. (canceled)
 17. The process of claim 13,wherein the eluate containing the recombinant therapeutic protein fromthe three of the four columns in the four-column PCCS is fed into thefourth column of the four-column PCCS.
 18. The process of claim 17,wherein the fourth column of the four-column PCCS performs the unitoperation of inactivating viruses by holding the eluate containingrecombinant therapeutic protein at a low pH for viral inactivation. 19.(canceled)
 20. The process of claim 18, wherein the MCCS2 is a secondperiodic counter current (PCCS2) chromatography system.
 21. The processof claim 20, further comprising adjusting the pH of the eluate from thefourth column of the four-column PCCS using an in-line buffer adjustmentreservoir before the eluate from the fourth column of the four-columnPCCS is fed into the PCCS2.
 22. The process of claim 21, where the PCCS2chromatography system comprises a three chromatography columns and achromatographic membrane.
 23. The process of claim 22, wherein the threechromatography columns in the PCCS2 perform the unit operation ofpurifying the recombinant therapeutic protein from the eluate of thePCCS1 through cation or anion exchange chromatography.
 24. The processof claim 23, wherein the eluate from the three chromatography columns inthe PCCS2 is fed into the chromatographic membrane in the PCCS2.
 25. Theprocess of claim 24, wherein the chromatographic membrane in the PCCS2performs the unit function of polishing the recombinant therapeuticprotein present in the eluate from the three chromatography columns inthe PCCS2 through cation or anion exchange chromatography. 26.(canceled)
 27. The process of claim 25, wherein the flow through andwash of the chromatographic membrane is the therapeutic protein drugsubstance.
 28. The process of claim 1, further comprising formulatingthe therapeutic protein drug substance into a pharmaceuticalcomposition. 29.-33. (canceled)
 34. An integrated and continuous processfor manufacturing a therapeutic protein drug substance comprising: (i)culturing mammalian cells that secrete a recombinant therapeutic proteinin a perfusion bioreactor that contains a liquid culture medium, whereina volume of the liquid culture medium that is substantially free ofcells is continuously or periodically removed from the perfusionbioreactor and fed into a first multi-column chromatography system(MCCS1); (ii) capturing said recombinant therapeutic protein in theremoved liquid culture medium using the MCCS1, wherein the eluate of theMCCS1 containing the recombinant therapeutic protein is continuously fedinto a second multi-column chromatography system (MCCS2); and (iii)purifying and polishing the therapeutic recombinant protein in theeluate of the MCCS1 using the MCCS2, wherein the eluate from the MCCS2is a therapeutic protein drug substance; wherein the process isintegrated and runs continuously from said removed liquid culture mediumto the eluate from the MCCS2 that is the therapeutic protein drugsubstance. 35.-64. (canceled)
 65. A biological manufacturing system,comprising: a first multi-column chromatography system (MCCS) comprisingan inlet; and a second MCCS comprising an outlet, wherein the first andsecond MCCSs are in fluid communication with each other, and wherein themanufacturing system is configured such that fluid is passed into theinlet, through the first and second MCCSs, and exits the manufacturingsystem through the outlet.
 66. The system of claim 65, furthercomprising a bioreactor, wherein the bioreactor and the inlet are influid communication with each other, and wherein the manufacturingsystem is configured such that fluid present in the bioreactor can bepassed into the inlet.
 67. The system of claim 65, wherein the firstMCCS or the second MCCS, or both, is/are configured to perform at leasttwo separate unit operations.
 68. The system of claim 65, wherein theuse of the MCCS1 or the MCCS2, or both, involves column switching. 69.The system of claim 67, wherein the first MCCS is configured to performthe unit operations of capturing the recombinant therapeutic protein andinactivating viruses.
 70. The system of claim 67, wherein the secondMCCS is configured to perform the unit operations of purifying andpolishing the recombinant therapeutic protein. 71.-73. (canceled) 74.The system of claim 65, wherein the first MCCS is a first periodiccounter current chromatography system (PCCS1).
 75. The system of claim74, wherein the PCCS1 comprises a four-column PCCS. 76.-91. (canceled)92. The system of claim 65, further comprising a pump system that is influid communication with the inlet. 93.-106. (canceled)
 107. Abiological manufacturing system, comprising two or more subsystems,wherein the two or more subsystems each comprise: (i) a firstmulti-column chromatography system (MCCS) comprising an inlet; and (ii)a second MCCS comprising an outlet, wherein the first and second MCCSsare in fluid communication with each other, and wherein themanufacturing system is configured such that fluid can be passed intothe inlet, through the first and second MCCS, and exit the manufacturingsystem through the outlet; wherein the two or more subsystems areconfigured such that they are each in fluid communication with a singlereservoir containing a fluid, and the fluid from the single reservoirpasses into the inlet of the two or more subsystems. 108.-111.(canceled)